Variant nucleoside-5′-phosphate producing enzyme

ABSTRACT

There is provided a mutant nucleoside-5′-phosphate producing enzyme with improved nucleoside-5′-phosphate producing ability of a nucleoside-5′-phosphate producing enzyme that has transphosphorylation activity and/or phosphatase activity and has one Lys residue, two Arg residues and two His residues with distances between their Cα&#39;s within a particular ranges and a space around them allowing a binding of a nucleoside, of which mutation is designed by using the crystal structure of EB-AP.

TECHNICAL FIELD

The present invention relates to mutant nucleoside-5′-phosphate producing enzymes with improved nucleoside-5′-phosphate producing ability, and a method for producing them. The present invention further relates to enzymes useful for the production of the aforementioned enzymes. Furthermore, the present invention relates to methods for producing a nucleoside-5′-phosphate as well as genes coding for the aforementioned mutant enzymes, recombinant DNA containing the genes and microorganisms that harbor the recombinant DNA, which are useful for the production methods. Nucleoside-5′-phosphates are useful as seasonings, drugs, raw materials therefor and so forth. The present invention is based on successful elucidation of the three-dimensional structures of a novel protein, and the three-dimensional structures have expandability that they are not limited to microorganisms.

BACKGROUND ART

As a method for efficiently producing nucleoside-5′-phosphates at a low cost by biochemically phosphorylating nucleosides, there was developed a method of efficiently producing a nucleoside-5′-phosphate without generation of byproducts of nucleoside-2′-phosphate acid and nucleoside-3′-phosphate isomers, which comprises allowing cells of a particular microorganism to act on a nucleoside and a phosphate donor selected from the group consisting of polyphosphoric acid (salt thereof), phenyl phosphate (salt thereof) and carbamyl phosphate under an acidic condition (Japanese Patent Laid-open (Kokai) No. 7-231793/1995).

Then, it was confirmed that the productivity of nucleoside-5′-phosphates can further be improved by obtaining a gene coding for an acid phosphatase from Escherichia blattae or Morganella morganii and expressing the gene in Escherichia coli in a large scale using genetic engineering techniques.

The structure of the acid phosphatase is shown in FIG. 2. That is, FIG. 2 shows alignment of the amino acid sequence of the acid phosphatase derived from Escherichia blattae (abbreviated as “EB-AP” hereinafter) with the amino acid sequences of acid phosphatases derived from Morganella morganii, Salmonella typhimurium and Zymomonas mobilis. The asterisks indicate conserved residues. The regions of the secondary structure are indicated with bars over the aligned sequences. The boxed portions are motives commonly observed in the acid phosphatase family. The motives consist of three domains, 1) KXXXXXXRP (SEQ ID NO: 121), 2) PSGH (SEQ ID NO: 122) and 3) SRXXXXXHXXXD (SEQ ID NO: 123). In these motives, X represents an arbitrary amino acid residue.

Although these acid phosphatases (FIG. 2) have transphosphorylation activity, they suffer from a drawback that their phosphatase activity for decomposing nucleoside-5′-phosphate into a nucleoside is dominant in wild-type strains, and hence accumulated nucleoside-5′-phosphate will be decomposed. Therefore, a large number of mutant enzymes were generated by the random mutagenesis method, and a mutant acid phosphatase having relatively improved transphosphorylation activity compared with phosphatase activity was found among the mutant enzymes. And it was demonstrated that the productivity of nucleoside-5′-phosphate was drastically improved by abundantly expressing a gene coding for the mutant acid phosphatase gene (Japanese Patent Laid-open (Kokai) No. 8-535568/1996).

This mutant acid phosphatase has improved affinity for a nucleoside, and it is thought that the transphosphorylation activity was improved by the enhanced affinity.

It was demonstrated that the aforementioned mutant acid phosphatase derived from Escherichia blattae (=G74D/I153T mutant enzyme (mutant enzyme in which 74th Gly is replaced with Asp and 153rd Ile is replaced with Thr, the mutation pattern will be similarly represented hereinafter)) showed weaker transphosphorylation activity compared with a corresponding G72D/I151T mutant enzyme of acid phosphatase derived from Morganella morganii (MM-AP), whereas a 10-residue replaced L63Q/A65Q/E66A/N69D/S71A/S72A/G74D/T135K/E136D/I153T mutant enzyme (referred to simply as “10-residue replaced mutant EB-AP” hereinafter), in which 8 amino acid residues were replaced with the amino acids of MM-AP that correspond on the primary structure basis, showed transphosphorylation activity substantially comparable to that of G72D/I151T mutant MM-AP (Japanese Patent Laid-open (Kokai) No. 9-161674/1997).

A method for producing a nucleoside-5′-phosphate has been established by expressing a large amount of the aforementioned G74D/I153T mutant enzyme gene, or the 10-residue replaced mutant enzyme gene in Escherichia coli (Japanese Patent Laid-open (Kokai) Nos. 9-37785/1997 and 10-201481/1999). However, a mutant EB-AP with further improved productivity is still desired.

DISCLOSURE OF THE INVENTION

The object of the present invention is to further improve the productivity of nucleoside-5′-phosphate by designing a mutant EB-AP based on the three-dimensional structure of EB-AP.

That is, aspects of the present invention are as follows:

(1) A mutant nucleoside-5′-phosphate producing enzyme with improved nucleoside-5′-phosphate producing ability, which is obtained by modifying a nucleoside-5′-phosphate producing enzyme that has transphosphorylation activity and/or phosphatase activity and has one Lys residue, two Arg residues and two His residues with distances between their Cα's within the ranges shown in FIG. 1 and a space around them allowing a binding of a nucleoside.

(2) The mutant nucleoside-5′-phosphate producing enzyme according to (1), wherein the distances between Cα's of the Lys residue, two Arg residues and two His residues are within the ranges shown in FIG. 1.

(3) The mutant nucleoside-5′-phosphate producing enzyme according to (1), wherein the nucleoside-5′-phosphate producing ability is improved by predicting a binding mode of the enzyme to a nucleoside such as inosine and guanosine and phosphorylated compounds thereof based on the atomic coordinate data obtained by X-ray crystallographic analysis of acid phosphatase derived from Escherichia blattae, and substituting, adding or deleting at least one amino acid residue and/or a prosthetic factor etc.

(4) The mutant nucleoside-5′-phosphate producing enzyme according to (1), wherein the enzyme is derived from a bacterium belonging to the genus Escherichia, Morganella, Providencia, Enterobacter or Klebsiella.

(5) A mutant nucleoside-5′-phosphate producing enzyme with improved nucleoside-5′-phosphate producing ability, which has an amino acid sequence of the acid phosphatase derived from Escherichia blattae including modification at one or more of the following positions (Ser72 or residues located within a distance of 10 Å from Ser72): 16, 67–76, 78–79, 96, 99–100, 102–104, 106–108, 149–154, 157, 179 and 183.

(6) A mutant nucleoside-5′-phosphate producing enzyme with improved nucleoside-5′-phosphate producing ability, which has transphosphorylation activity and/or phosphatase activity, and has modification at one or more of positions corresponding to the following positions in the amino acid sequence of the acid phosphatase derived from Escherichia blattae (Ser72 of Escherichia blattae acid phosphatase or residues located within a distance of 10 Å from Ser72): 16, 67–76, 78–79, 96, 99–100, 102–104, 106–108, 149–154, 157, 179 and 183 in amino acid sequence alignment with the acid phosphatase derived from Escherichia blattae.

(7) A mutant nucleoside-5′-phosphate producing enzyme with improved nucleoside-5′-phosphate producing ability, which has transphosphorylation activity and/or phosphatase activity, and has modification at one or more of positions corresponding to the following positions in the amino acid sequence of the acid phosphatase derived from Escherichia blattae (Ser72 of Escherichia blattae acid phosphatase or residues located within a distance of 10 Å from Ser72): 16, 67–76, 78–79, 96, 99–100, 102–104, 106–108, 149–154, 157, 179 and 183 in alignment with the three-dimensional structure of the acid phosphatase derived from Escherichia blattae performed by the threading method.

(8) The mutant nucleoside-5′-phosphate producing enzyme according to (6), wherein the three-dimensional structure of the enzyme is put close to that of an enzyme derived from another organism having transphosphorylation activity higher than that of a wild-type of the enzyme having the transphosphorylation activity and/or phosphatase activity by making modification at one or more positions other than the positions (Ser72 of Escherichia blattae acid phosphatase or residues present within a distance of 10 Å from Ser72).

(9) A mutant nucleoside-5′-phosphate producing enzyme with improved nucleoside-5′-phosphate producing ability, which has an amino acid sequence of the acid phosphatase derived from Escherichia blattae including modification at one or more of the following positions: 16, 71, 72, 73, 103, 104, 140, 151 and 153.

(10) A mutant nucleoside-5′-phosphate producing enzyme with improved nucleoside-5′-phosphate producing ability, which has transphosphorylation activity and/or phosphatase activity, and has modification at one or more of positions corresponding to the following positions of the amino acid sequence of the acid phosphatase derived from Escherichia blattae: 16, 71, 72, 73, 103, 104, 140, 151 and 153 in amino acid sequence alignment with the acid phosphatase derived from Escherichia blattae.

(11) A mutant nucleoside-5′-phosphate producing enzyme with improved nucleoside-5′-phosphate producing ability, which has transphosphorylation activity and/or phosphatase activity, and has modification at one or more of positions corresponding to the following positions of the amino acid sequence of the acid phosphatase derived from Escherichia blattae: 16, 71, 72, 73, 103, 104, 140, 151 and 153 in alignment with the three-dimensional structure of the acid phosphatase derived from Escherichia blattae performed by the threading method.

(12) A mutant nucleoside-5′-phosphate producing enzyme, which has an amino acid sequence of the acid phosphatase derived from Escherichia blattae including replacement of the 72nd residue with another amino acid residue.

(13) A mutant nucleoside-5′-phosphate producing enzyme, which has transphosphorylation activity and/or phosphatase activity, and has replacement of a residue corresponding to the 72nd residue of the amino acid sequence of the acid phosphatase derived from Escherichia blattae with another amino acid in amino acid sequence alignment with the acid phosphatase derived from Escherichia blattae.

(14) A mutant nucleoside-5′-phosphate producing enzyme, which has transphosphorylation activity and/or phosphatase activity, and has replacement of a residue corresponding to the 72nd residue of the amino acid sequence of the acid phosphatase derived from Escherichia blattae with another amino acid in alignment with the three-dimensional structure of the acid phosphatase derived from Escherichia blattae performed by the threading method.

(15) The mutant nucleoside-5′-phosphate producing enzyme according to (10), wherein the enzyme is derived from Enterobacter aerogenes, and the amino acid sequence of the enzyme includes replacement with another amino acid residue of at least one amino acid residue among the 14th leucine residue, the 61st leucine residue, the 63rd alanine residue, the 64th glutamic acid residue, the 67th asparagine residue, the 69th serine residue, the 70th alanine residue, the 71st glycine residue, the 72nd glycine residue, the 101st isoleucine residue, the 102nd glutamic acid residue, the 133rd threonine residue, the 134th glutamic acid residue, the 138th leucine residue, 149th threonine residue and the 151st isoleucine residue.

(16) The mutant nucleoside-5′-phosphate producing enzyme according to (12), wherein the enzyme is derived from Enterobacter aerogenes, and the enzyme has any one of following amino acid replacements:

-   -   (a) mutation consisting of replacements of the 61st leucine         residue with a glutamine residue, the 63rd alanine residue with         a glutamine residue, the 64th glutamic acid residue with an         alanine residue, the 67th asparagine residue with an aspartic         acid residue, the 69th serine residue with an alanine residue,         the 70th alanine residue with a valine residue, the 72nd glycine         residue with an aspartic acid residue, the 102nd glutamic acid         residue with a leucine residue, the 133rd threonine residue with         a lysine residue, the 134th glutamic acid residue with an         aspartic acid residue, the 149th threonine residue with a serine         residue and the 151st isoleucine residue with a serine residue;     -   (b) mutation consisting of replacements of the 61st leucine         residue with a glutamine residue, the 63rd alanine residue with         a glutamine residue, the 64th glutamic acid residue with an         alanine residue, the 67th asparagine residue with an aspartic         acid residue, the 69th serine residue with an alanine residue,         the 70th alanine residue with a valine residue, the 72nd glycine         residue with an aspartic acid residue, the 133rd threonine         residue with a lysine residue, the 134th glutamic acid residue         with an aspartic acid residue, the 149th threonine residue with         a alanine residue and the 151st isoleucine residue with a serine         residue;     -   (c) mutation consisting of replacements of the 61st leucine         residue with a glutamine residue, the 63rd alanine residue with         a glutamine residue, the 64th glutamic acid residue with an         alanine residue, the 67th asparagine residue with an aspartic         acid residue, the 69th serine residue with an alanine residue,         the 70th alanine residue with a glutamic acid residue, the 72nd         glycine residue with an aspartic acid residue, the 133rd         threonine residue with a lysine residue, the 134th glutamic acid         residue with an aspartic acid residue, the 149th threonine         residue with a glycine residue and the 151st isoleucine residue         with a serine residue;     -   (d) mutation consisting of replacements of the 61st leucine         residue with a glutamine residue, the 63rd alanine residue with         a glutamine residue, the 64th glutamic acid residue with an         alanine residue, the 67th asparagine residue with an aspartic         acid residue, the 69th serine residue with an alanine residue,         the 70th alanine residue with a lysine residue, the 72nd glycine         residue with an aspartic acid residue, the 133rd threonine         residue with a lysine residue, the 134th glutamic acid residue         with an aspartic acid residue, the 149th threonine residue with         a glycine residue and the 151st isoleucine residue with a serine         residue; and     -   (e) mutation consisting of replacements of the 61st leucine         residue with a glutamine residue, the 63rd alanine residue with         a glutamine residue, the 64th glutamic acid residue with an         alanine residue, the 67th asparagine residue with an aspartic         acid residue, the 69th serine residue with an alanine residue,         the 70th alanine residue with a methionine residue, the 72nd         glycine residue with an aspartic acid residue, the 102nd         glutamic acid residue with a glutamine residue, the 133rd         threonine residue with a lysine residue, the 134th glutamic acid         residue with an aspartic acid residue, the 149th threonine         residue with a serine residue and the 151st isoleucine residue         with a serine residue.

(17) A method for producing a mutant nucleoside-5′-phosphate producing enzyme, wherein a mutant enzyme with improved nucleoside-5′-phosphate producing ability is produced by substituting, adding or deleting at least one amino acid residue in the active site of an enzyme having transphosphorylation activity and/or phosphatase activity and/or an amino acid residue located within a distance of 10 Å from the active site, in which the active site is determined based on the three-dimensional structure of the enzyme obtained by X-ray crystallographic analysis of the enzyme or a complex of the enzyme with molybdate.

(18) A method for producing an inhibitor for a phosphatase or transphosphorylation enzyme, which utilizes the atomic coordinates of the acid phosphatase derived from Escherichia blattae.

(19) A crystal of an enzyme having transphosphorylation activity and/or phosphatase activity or the complex of the enzyme with molybdate.

(20) A crystal of acid phosphatase derived from Escherichia blattae, which has a space group P6₃22 of a hexagonal system.

(21) A crystal of mutant enzyme acid phosphatase G74D/I153T derived from Escherichia blattae, which has a space group P2₁2₁2₁ of a rhombic system.

(22) A crystal of complex of acid phosphatase derived from Escherichia blattae and molybdate (reaction intermediate analogue), which has a space group P3₁21 of a trigonal system.

(23) A gene coding for any one of the enzymes according to any one of (1) to (16).

(24) A recombinant DNA, which contains the gene according to (23).

(25) A microorganism, which contains the gene according to (23) or the recombinant DNA according to (24).

(26) A method for producing a nucleoside-5′-phosphate, which comprises allowing the enzyme according to any one of (1) to (16), a microorganism containing it or the microorganism according to (25) to act on a nucleoside and a phosphate donor to produce nucleoside-5′-phosphate and collecting it.

(27) The method according to (26), wherein the enzyme, the microorganism containing it or the microorganism according to (25) is allowed to act on a nucleoside and a phosphate donor under a condition of pH 3.0–5.5.

The present invention provides a method for producing nucleoside-5′-phosphate using a mutant EB-AP, which is designed based on a binding model of EB-AP with a nucleoside constructed on the basis of the three-dimensional structure of EB-AP.

However, the mutant nucleoside-5′-phosphate producing enzyme of the present invention does not include the G74D/I153T mutant EB-AP, the G72D/I151T mutant MM-AP and the 10-residue replaced mutant EB-AP.

The present invention will be specifically explained hereinafter.

(1) In order to determine the three-dimensional structure of a protein by X-ray crystallography, the protein must be crystallized (details will be described in Examples 1–3). In order to crystallize a protein, many parameters must be determined on the trial and error basis, such as pH, kind of buffer, concentration of buffer, kind of precipitating agent, concentration of precipitating agent, concentrations of additives such as metals, concentration of protein and purity of protein. Therefore, it usually takes several months to several years before obtaining crystals, and there is often a case where crystals cannot be obtained in spite of such great effort. While crystallization is indispensable for the determination of the three-dimensional structure, it is also industrially useful as purification method of proteins to high purity, method for storage of proteins with high density and high protease resistance as well as a process prior to utilization as immobilized enzymes.

(2) By irradiating the produced crystals with an x-ray, diffraction data are collected. A protein crystal is often damaged by X-ray irradiation, and thus suffers from degradation of diffraction ability. For such a case, low temperature measurement recently becomes popular, in which crystals are rapidly cooled to about −173° C. and diffraction data are collected in that state. Upon cooling, it is necessary to devise a solvent composition so that the crystals should not be destroyed and the whole system should be vitrified.

(3) In order to perform crystal structure analysis, phase information is required in addition to the diffraction data.

Since three-dimensional structures of proteins analogous to EB-AP have not been known yet, the problem concerning the phase must be solved by the heavy atom isomorphous replacement method. The heavy atom isomorphous replacement method is a method for obtaining phase information by introducing a metal of a large atomic number such as mercury and platinum into a crystal and utilizing contribution of strong x-ray scattering ability of metal atoms to X-ray diffraction data to obtain phase information. If the three-dimensional structure of wild-type EB-AP is once determined, crystal structures of analogous compounds thereof such as mutant enzymes and reaction intermediate analogues can be determined by the molecule replacement method by using it. The molecule replacement method is a technique for crystal structure determination by using a three-dimensional structure of a protein analogous to the protein of which crystal structure is to be determined, when the three-dimensional structure of the analogous protein is known. For example, if a three-dimensional structure of a wild-type of a certain protein is known, the molecule replacement method can be used for the determination of a crystal structure of a mutant protein or a chemically modified protein thereof.

As for the G74D/I153T mutant EB-AP, its crystal structure is determined for elucidating the molecular mechanism for the enhancement of affinity for a nucleoside by substitution of two amino acids.

As for a reaction intermediate analogue, its crystal structure is determined in order to construct its binding model with a nucleoside. A nucleoside binds to EB-AP to which phosphate group to be donated binds through a covalent bond, i.e., a reaction intermediate, and then converted into a nucleoside-5′-phosphate. Since the reaction intermediate of EB-AP is unstable, its structure cannot be determined. It was considered that, however, since a reaction intermediate analogue to which molybdate binds through a covalent bond instead of phosphate was not hydrolyzed, its structure could be determined. The details will be described in Examples 4, 6, and 7.

(4) On computer graphics (CG), a binding mode model is constructed by fitting a nucleoside to a hollow near the molybdate binding site based on the binding site in the three-dimensional structure of the reaction intermediate analogue (FIG. 3). For the construction of the model, a program such as QUANTA and INSIGHT II of MSI (United States) is utilized.

FIG. 3 is a photograph of the crystal structure of the aforementioned binding mode model.

The details will be described in Examples 5 and 8.

(5) By extensively examining the binding model, a mutation that is considered to increase the affinity for a nucleoside is designed. In order to improve the affinity, it is effective to enhance hydrophobic interaction, electrostatic interaction, hydrogen bond, π—π interaction (interaction of magnetic fields generated by ring currents of aromatic rings) and CH/π interaction (interaction of magnetic field generated by ring current of an aromatic ring and electrons of methyl group).

For example, since it is expected that Ser72 most strongly interacts with a base of nucleoside in the binding mode model, it can be considered that replacement of the residue with Phe, Tyr and Trp enhances the hydrophobic interaction and the π—π interaction, replacement with Val, Ile and Leu enhances the hydrophobic interaction and the CH/π interaction, and replacement with Glu and Asp enhances the electrostatic interaction and the hydrogen bond. Further, the hydrophobic interaction and so forth may also be enhanced by replacement with other amino acids, in particular, replacement with an amino acid having a longer side chain.

It is also expected that replacements of Leu16, Ser71, Ser73 and Glu104 with Phe, Tyr and Trp generate the π—π interaction between the aromatic rings of the replaced amino acid residues and a base of nucleoside. Further, it is expected that replacements of Ile103 or Thr153 with a hydrophilic residue having a longer chain generate a hydrogen bond with the ribose of nucleoside. Furthermore, it is expected that replacement of Thr151 that is present near the nucleoside binding site and buried inside the protein with an amino acid residue having a small side chain such as Ser, Ala and Gly produces a space in the protein so that flexibility of the nucleoside binding site is increased and the binding site can have a conformation more suitable for binding with a nucleoside. Although Leu410 is separated from Ser72 by more than 10 Å, it is located near the phosphate binding site in the three-dimensional structure of a reaction intermediate analogue. Therefore, it is considered that if this residue is replaced, the structure around the phosphate binding site of the reaction intermediate is changed, and as a result, it also influences the structure of the nucleoside binding site and fluctuation thereof. It is expected that if this residue is replaced with more bulky Phe, positively charged Lys or negatively charged Glu, the affinity with a nucleoside may be changed.

The aforementioned mutations are introduced into, for example, the G74D/I153T mutant EB-AP. However, the mutant enzyme to which the mutations are introduced is not limited to the G74D/I153T mutant EB-AP. For example, it is also possible to introduce the mutations into the 10-residue replaced mutant EB-AP.

The details will be described in Example 10.

(6) A plasmid containing a gene coding for a mutant EB-AP is produced. The gene coding for a mutant EB-AP can be obtained by introducing a target mutation into a gene coding for a wild-type EB-AP through site-specific mutagenesis.

The plasmid containing the gene coding for a mutant EB-AP is introduced into Escherichia coli JM109 to produce a mutant EB-AP. A Km value, which serves as an index of affinity of the mutant EB-AP for inosine, and transphosphorylation activity to convert inosine into 5′-inosinic acid are measured to evaluate the performance of the mutant EB-AP. It is considered that the production amount of nucleoside-5′-phosphate much depends on the Km value. After pyrophosphoric acid reacts with EB-AP and a phosphate ion dissociates so that a reaction intermediate with the phosphate group covalently bonded to EB-AP is formed, if a water molecule attacks it, the phosphate group will be released (phosphatase reaction). It means that the pyrophosphoric acid was consumed vainly without generating nucleoside-5′-phosphate. On the other hand, if a nucleoside attacks the reaction intermediate, the phosphate group will form a phosphomonoester linkage with the nucleoside, and the generated nucleoside-5′-phosphate will dissociate from EB-AP (transphosphorylation reaction). It means that the pyrophosphoric acid was utilized for the production of nucleoside-5′-phosphate. That is, if water binds to the reaction intermediate, phosphatase activity will be exerted, and if the nucleoside binds to the intermediate, transphosphorylation activity will be exerted. If the affinity of EB-AP for the nucleoside is increased, i.e., if the Km value is decreased, the likelihood of the transphosphorylation reaction will become high. Further, if the hydrophobicity around the phosphate binding site is increased and thus it becomes difficult for water to approach the binding site, the phosphatase activity will become weaker and the transphosphorylation activity will become relatively stronger. Thus, the expression of “nucleoside-5′-phosphate producing ability is improved”used for the present invention may mean either improvement of the transphosphorylation activity or decrease of the phosphatase activity of a mutant acid phosphatase (see WO96/37603), or it may mean the both.

The details will be described in Example 11.

(7) By using Escherichia coli JM109 transformed with the plasmid containing a mutant EB-AP gene, in which the Km value is decreased and the transphosphorylation activity is increased, experimental production of 5′-inosinic acid from inosine is performed. The reaction is performed at 30° C. for 45 hours, and the variation with time in the production amount of 5′-inosinic acid is monitored. The details will be described in Example 13.

If mutation sites that decrease Km are found out, a mutant enzyme having improved affinity for a nucleoside and higher productivity can be produced by combining two or more mutations. By repeating the site-specific mutagenesis, two or more mutation sites can be cumulatively introduced. Further, upon introduction of site-specific mutations, if primers having mixed nucleotides as the nucleotides of a portion coding for an amino acid residue for which the mutation is introduced, a library of mutant genes in which a particular encoded amino acid is replaced with all of amino acid residues can be produced. If a mutation is introduced into multiple sites by using primers having mixed nucleotides, a library of mutant genes coding for extremely many kinds of mutant enzymes can be produced. It is also effective to introduce a library constructed as described above into Escherichia coli, and select a mutant comprising a combination of amino acid substitutions providing high activity from the expressed library.

Other than EB-AP, an enzyme having an active site similar to that of EB-AP and a space for binding of a nucleoside may potentially be utilized for the production of nucleoside-5′-phosphate. The active site must have amino acid residues indispensable for the activity, and the residues must be configured in a suitable relationship in special positions. In EB-AP, Lys115, Arg122, His150, Arg183 and His189 are indispensable for the activity, and it is possible to define the relationship of special positions with distances between Cα's of these 5 residues. In the present invention, since crystal structures of three kinds of EB-AP, wild-type, G74D/I153T mutant and reaction intermediate analogue, were determined, Ca interatomic distances of the active residues in each structure were measured, and summarized in Table 1. Since the distance distribution for each of the distances shown in Table 1 showed a range of about 1 Å, it was considered that the desired active site could be formed if each distance is in the range of from a distance shorter than the shortest distance by 1 Å (Table 1, lower limit) to a distance longer than the longest distance by 1 Å (Table 1, upper limit). In FIG. 1, positional relationship of the five residues is shown with upper limits and lower limits of the distances between Cα's.

In Example 8, it will be demonstrated that, in MM-AP, which is an analogous enzyme of EB-AP, all the interatomic distances between active residues fall within the ranges defined based on the three-dimensional structure of EB-AP. In that example, although not a wild-type, but the G72D/I151T mutant was exemplified, it is considered that there is not significant difference in the active site structure between a wild-type and a mutant of the same enzyme. This assumption is supported by the fact that the structures of the active sites in a wild-type and the G74D/I153T mutant of EB-AP are basically identical to each other (see Table 1).

TABLE 1 G74D/ Reaction Wild- I153T intermediate Lower Upper type mutant analogue limit limit Lys115 Arg122 11.6 Å 11.6 Å 11.4 Å 10.4 Å 12.6 Å His150 12.4 Å 12.3 Å 12.8 Å 11.3 Å 13.8 Å Arg183 16.4 Å 16.3 Å 15.5 Å 14.5 Å 17.4 Å His189 12.6 Å 12.1 Å 11.7 Å 10.7 Å 13.6 Å Arg122 His150 13.2 Å 13.6 Å 14.2 Å 12.2 Å 15.2 Å Arg183 10.4 Å 10.5 Å 10.8 Å  9.4 Å 11.8 Å His189  5.6 Å  5.5 Å  5.7 Å  4.5 Å  6.7 Å His150 Arg183  8.4 Å  8.8 Å  7.7 Å  6.7 Å  9.8 Å His189  9.8 Å 10.0 Å 10.0 Å  8.8 Å 11.0 Å Arg183 His189  5.5 Å  5.8 Å  5.7 Å  4.5 Å  6.8 Å

In order to convert a nucleoside into nucleoside-5′-phosphate by phosphorylation, the presence of the active site that consists of the aforementioned five residues alone is not sufficient, and a nucleoside must be able to bind to a suitable position. In EB-AP, in the neighborhood of phosphate group, a slot-like space suitable for binding of a nucleoside is present on the surface of the molecule [(FIG. 3): it can be displayed on computer graphics (CG) by using the atomic coordinates represented in the appended FIGS. 10–45]. This slot is defined as a space surrounded by four residues of Leu16, Ser72, Glu104 and His189. Even if an enzyme has the active site, the enzyme is unsuitable as a nucleoside-5′-phosphate producing enzyme without a suitable space for binding of a nucleoside.

The mutant nucleoside-5′-phosphate producing enzyme of the present invention can be obtained by modifying an enzyme having the transphosphorylation activity and/or the phosphatase activity, in which the distances between Cα's of five residues of Lys115, Arg122, His150, Arg183 and His189 are within the ranges shown in FIG. 1. However, so long as it has a space for binding of a nucleoside near the aforementioned five residues and has transphosphorylation activity, the distances between Cα's of the five residues may not be within the ranges shown in FIG. 1. Nonetheless, the mutant acid phosphatase to be obtained is preferably has the distances between Cα's of the aforementioned five residues falling within the ranges shown in FIG. 1.

The present invention further provides a mutant EB-AP comprising a replacement of Ser72 with another amino acid, preferably with any one of Phe, Tyr, Trp, Val, Leu, Glu, Asp, Gln, Met, Thr, Arg and Lys, and, in addition, two mutations of G74D and I153T, which are already published as mutations enhancing the nucleoside-5′-phosphate producing ability (henceforth referred to as the “3-residue replaced mutant EB-AP”). Furthermore, the residues present within a distance of 10 Å from Ser72 (residue numbers: 16, 70–71, 73–76, 100, 102–104, 106–108, 115, 148–154, 183) are very likely to interact with a nucleoside, and the present invention also provides a mutant EB-AP comprising one or more replacements of these amino acid residues with one or more other amino acids. The term “replacement (substitution)” used herein include not only an artificial replacement of amino acid residue, but also selection of another acid phosphatase belonging to the same enzyme family as EB-AP and comprising a naturally occurring replacement. However, the present invention is still able to provide a mutant EB-AP containing a mutation point other than the aforementioned amino acid residues.

Further, another acid phosphatase belonging to the same enzyme family as EB-AP may be used for the production of nucleoside-5′-phosphate, if an amino acid mutation homologous to those used for EB-AP is made in that enzyme. As such an acid phosphatase, there can be mentioned those enzymes derived from Escherichia bacteria other than Escherichia blattae, Morganella bacteria other than Morganella morganii, Providencia bacteria such as Providencia stuartii, Enterobacter bacteria, Klebsiella bacteria such as Klebsiella planticola and so forth. However, the amino acid residues of EB-AP do not necessarily correspond to the amino acid residues of the same numbers in other acid phosphatases. For example, Ser72 of EB-AP corresponds to Ala70 in MM-AP. The correspondence of amino acid residues in two different proteins can be determined by alignment of the amino acid sequences (Sequence Alignment) when the identity between the both amino acid sequences is about 20% or more, or alignment of the three-dimensional structure and amino acid sequence (Threading) when the identity between the both amino acid sequences is about 20% or less. The former can be performed with a program of BLAST etc., and the latter can be performed with a program of INSIGHT II etc.

The amino acid sequence alignment of EB-AP and a wild-type acid phosphatase (wild-type EA-AP) derived from Enterobacter aerogenes obtained by using BLAST is described in Example 20 (see FIG. 9). Further, alignment of the amino acid sequences of EB-AP, MM-AP and acid phosphatases derived from Salmonella typhimurium and Zymomonas mobilis is shown in FIG. 2. The sequences of the genes coding for EB-AP, MM-AP, acid phosphatases derived from Salmonella typhimurium and Zymomonas mobilis and EA-AP and amino acid sequences thereof are shown in SEQ ID NOS: 1-10. Among these sequences, EA-AP is of 10-residue replaced mutant, and others are of wild-type. Further, EA-AP is represented as a proprotein containing a signal peptide. In addition, the amino acid sequences of the acid phosphatase derived from Providencia stuartii (GenBank accession X64820) and Klebsiella planticola (GenBank accession E16588) and the nucleotide sequences of the genes coding for them are known.

When acid phosphatase is produced as a precursor protein having a signal peptide and then processed into a mature protein through removal of the signal peptide, alignment is performed for the amino acid sequence of the mature protein.

As for BLAST, a file compatible with a computer to be used can be obtained from the files stored in /blast/executable from the NCBI database managed by the National Library of Medicine, National Institutes of Health (Bethesda, Md. USA) by using an FTP server.

The details about the operation method are described in the installation and procedures packet available on the website for the National Institutes of Health (Bethesda, Md., USA).

While the improvement of nucleoside-5′-phosphate producing ability is often attained by enhancement of affinity for a nucleoside, besides it, it may also be attained by improvement in reaction rate, shift of optimum pH, enhancement of thermal stability etc. The shift of optimum pH can be attained by changing pK of active residues (Protein Engng., 11, 383–388 (1998)). The enhancement of thermal stability can be attained by introduction of proline residues, replacement of residues that show left-handed helical structure with glycine residues (Protein Engng., 6, 85–91 (1993)), filling space in the protein (Biochemistry, 32, 6171–6178 (1993)) and so forth.

Further, the mutant nucleoside-5′-phosphate producing enzyme of the present invention may have mutations of other amino acid residues in addition to the aforementioned mutations, so long as such mutations do not adversely affect the nucleoside-5′-phosphate producing ability. Examples of such mutations include a mutation that enhances temperature stability (refer to Japanese Patent Laid-open (Kokai) No. 10-201481/1998). In addition, a single mutation may exert the effect for improvement of the nucleoside-5′-phosphate producing ability and another effect, for example, for improvement of temperature stability. In any case, an enzyme containing such a mutation falls within the scope of the mutant nucleoside-5′-phosphate producing enzyme of the present invention, so long as its nucleoside-5′-phosphate producing ability is eventually enhanced.

For example, the nucleoside-5′-phosphate producing ability may be enhanced even by replacing a residue that is not Ser72 of EB-AP or is not present within a distance of 10 Å from the Ser72 with another residue. Examples of such a residue include Leu140 for EB-AP and Leu138 for EA-AP.

As described in detail above, the three-dimensional structure is effective for producing a mutant of which affinity for a nucleoside and nucleoside-5′-phosphate producing ability are improved. However, the three-dimensional structure is effective not only in changing the affinity for a nucleoside of an enzyme, but also in changing affinity for a phosphate donor. As disclosed in Japanese Patent Laid-open (Kokai) No. 9-37785/1997, the enzyme can use various phosphoric acid ester compounds such as polyphosphoric acid (salt thereof), phenyl phosphate (salt thereof), acetyl phosphate (salt thereof) and carbamyl phosphate (salt thereof) as a phosphate donor. However, it is also possible to broaden its substrate specificity for the phosphate donor, or improve the utilization factor of phosphate by designing a mutation that increases the affinity for a phosphoric acid ester compound in a manner similar to that for designing a mutation enhancing the affinity for a nucleoside.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the five amino acid residues serving as the components of the active site having the phosphatase activity or the transphosphorylation activity, and their relationship in special positions as distances between Cα atoms. The identified amino acids are as follows:

-   -   Lys: 115^(th) residue of SEQ ID NO: 124;     -   Arg (N-terminus side): 122^(nd) residue of SEQ ID NO: 124;     -   His (N-terminus side): 150^(th) residue of SEQ ID NO: 124;     -   Arg (C-terminus side): 183^(rd) residue of SEQ ID NO: 124; and     -   His (C-terminus side): 189^(th) residue of SEQ ID NO: 124.

FIG. 2 shows alignment of the amino acid sequence of EB-AP (Escherichia blattae-acid phosphatase, SEQ ID NO: 2) with the amino acid sequences of the acid phosphatases derived from Morganella morganii (SEQ ID NO: 4), Salmonella typhimurium (SEQ ID NO: 6), and Zymomonas mobilis (SEQ ID NO: 8).

FIG. 3 is a photograph of computer graphics (CG), which represents a crystal structure in a binding mode model of the EB-AP reaction intermediate analogue and inosine.

FIG. 4 is a photograph of CG, which shows a crystal structure of the hexamer molecule of EB-AP.

FIG. 5 is a photograph of CG, which shows a crystal structure of a subunit of EB-AP.

FIG. 6 shows a structure of the active site of EB-AP.

FIG. 7 shows primer sets used for site-directed mutagenesis.

FIG. 8 shows primer sets used for site-directed mutagenesis.

FIG. 9 shows the result of amino acid sequence alignment of EB-AP (SEQ ID NO: 124) and a wild-type acid phosphatase derived from Enterobacter aerogenes (wild-type EA-AP, SEQ ID NO: 125) performed by using the program BLAST.

FIG. 10 shows the crystallographic data (1) for the structure of EB-AP.

FIG. 11 shows the crystallographic data (2) for the structure of EB-AP.

FIG. 12 shows the crystallographic data (3) for the structure of EB-AP.

FIG. 13 shows the crystallographic data (4) for the structure of EB-AP.

FIG. 14 shows the crystallographic data (5) for the structure of EB-AP.

FIG. 15 shows the crystallographic data (6) for the structure of EB-AP.

FIG. 16 shows the crystallographic data (7) for the structure of EB-AP.

FIG. 17 shows the crystallographic data (8) for the structure of EB-AP.

FIG. 18 shows the crystallographic data (9) for the structure of EB-AP.

FIG. 19 shows the crystallographic data (10) for the structure of EB-AP.

FIG. 20 shows the crystallographic data (11) for the structure of EB-AP.

FIG. 21 shows the crystallographic data (12) for the structure of EB-AP.

FIG. 22 shows the crystallographic data (13) for the structure of EB-AP.

FIG. 23 shows the crystallographic data (14) for the structure of EB-AP.

FIG. 24 shows the crystallographic data (15) for the structure of EB-AP.

FIG. 25 shows the crystallographic data (16) for the structure of EB-AP.

FIG. 26 shows the crystallographic data (17) for the structure of EB-AP.

FIG. 27 shows the crystallographic data (18) for the structure of EB-AP.

FIG. 28 shows the crystallographic data (19) for the structure of EB-AP.

FIG. 29 shows the crystallographic data (20) for the structure of EB-AP.

FIG. 30 shows the crystallographic data (21) for the structure of EB-AP.

FIG. 31 shows the crystallographic data (22) for the structure of EB-AP.

FIG. 32 shows the crystallographic data (23) for the structure of EB-AP.

FIG. 33 shows the crystallographic data (24) for the structure of EB-AP.

FIG. 34 shows the crystallographic data (25) for the structure of EB-AP.

FIG. 35 shows the crystallographic data (26) for the structure of EB-AP.

FIG. 36 shows the crystallographic data (27) for the structure of EB-AP.

FIG. 37 shows the crystallographic data (28) for the structure of EB-AP.

FIG. 38 shows the crystallographic data (29) for the structure of EB-AP.

FIG. 39 shows the crystallographic data (30) for the structure of EB-AP.

FIG. 40 shows the crystallographic data (31) for the structure of EB-AP.

FIG. 41 shows the crystallographic data (32) for the structure of EB-AP.

FIG. 42 shows the crystallographic data (33) for the structure of EB-AP.

FIG. 43 shows the crystallographic data (34) for the structure of EB-AP.

FIG. 44 shows the crystallographic data (35) for the structure of EB-AP.

FIG. 45 shows the crystallographic data (36) for the structure of EB-AP.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained more specifically with reference to the following examples. However, the present invention is not limited to these examples.

EXAMPLE 1 Crystallization of Wild-Type EB-AP

Crystallization was performed by using vapor diffusion in the hanging drop method. A 20 mM sodium phosphate buffer (pH 8.0) containing the wild-type EB-AP (concentration: 10 mg/ml) and a 100 mM Tris-HCl buffer containing 45% (w/v) of polyethylene glycol 400 in the same amounts (7–10 μl each) were dropped and mixed on siliconized cover glass, and a well filled with 500 μl of 100 mM Tris-HCl buffer containing 45% (w/v) of polyethylene glycol 400 was covered with the cover glass so that a drop of the mixed solution should be hung above the well and left stand at 20° C. Crystals appeared within a few days, and they grew up to hexagonal columnar crystals in a measurable size (about 0.3×0.3×1.2 mm) within 1 to 2 weeks. For the X-ray data measurement, the crystals were transferred into 100 mM Tris-HCl buffer (pH 8.0) containing 50% (w/v) polyethylene glycol 400.

Upon handling of these crystals, it was necessary to pay attention to the following points. 1) Since the crystals were very likely to collapse by contacting with a container or tools when crystals were taken out from the droplet, the crystallization scheme of the sitting drop method could not be used (although crystals grew), the hanging drop method described herein was used. 2) Since crystals would degrade during measurement and hence resolution would be gradually reduced if the measurement was performed at room temperature, it was necessary to perform the measurement at a low temperature. The time required for mounting the crystals on a stage was made as short as possible to prevent them from being exposed to air.

Using an X-ray diffraction apparatus, R-AXIS IIc of Rigaku Co., Ltd., X-ray diffraction data were collected and crystallographic parameters were determined. The space group was determined to be P6₃22 and the cell parameters to be a=b=124.4 Å and c=97.7 Å. Assuming that one subunit having a molecular weight of 25000 is contained in an asymmetric unit, the solvent content of the crystals is determined to be 72%.

EXAMPLE 2 Crystallization of G74D/I153T Mutant EB-AP

Crystallization was performed by using vapor diffusion in the hanging drop method. A 20 mM sodium phosphate buffer (pH 8.0) containing the G74D/I153T mutant EB-AP (concentration: 20 mg/ml) and a 20 mM Tris-HCl buffer containing 38% (w/v) of polyethylene glycol 400 in the same amounts (5 μl each) were dropped and mixed on siliconized cover glass, and a well filled with 500 μl of 20 mM Tris-HCl buffer containing 38% (w/v) of polyethylene glycol 400 was covered with the cover glass so that a drop of the mixed solution should be hung above the well and left stand at 20° C. Crystals appeared within a few days, and they grew up to tabular crystals in a measurable size (about 0.7×0.4×0.2 mm) within 1 week. For the X-ray data measurement, the crystals were transferred into 100 mM Tris-HCl buffer (pH 8.0) containing 50% (w/v) polyethylene glycol 400.

Using an X-ray diffraction apparatus, R-AXIS IIc of Rigaku Co., Ltd., X-ray diffraction data were collected and crystallographic parameters were determined. The space group was determined to be P2₁2₁2₁ and the cell parameters to be a=138.0 Å, b=168.3 Å and c=58.2 Å. Assuming that one subunit having a molecular weight of 15000 is contained in an asymmetric unit, the solvent content of the crystals is determined to be 64%.

EXAMPLE 3 Crystallization of Complex of Wild-Type EB-AP and Molybdate (Reaction Intermediate Analogue)

Crystallization was performed by a co-crystallization method by using vapor diffusion in the sitting drop method. A 20 mM sodium phosphate buffer (pH 8.0) containing the wild-type EB-AP (concentration: 10 mg/ml) and a 100 mM Tris-HCl buffer containing 40% (w/v) of polyethylene glycol 400 and 1 mM sodium molybdate in the same amounts (15 μl each) were added dropwise to a hollow of a bridge provided on a well filled with 500 μl of 100 mM Tris-HCl buffer containing 40% (w/v) of polyethylene glycol 400, mixed, and left stand at 20° C. Crystals appeared within a few days, and they grew up to rhomboidal crystals in a measurable size (about 0.3×0.3×0.3 mm) within 1 to 2 weeks. For the X-ray data measurement, the crystals were transferred into 100 mM Tris-HCl buffer (pH 8.0) containing 50% (w/v) polyethylene glycol 400.

Using an X-ray diffraction apparatus, R-AXIS IIc of Rigaku Co., Ltd., X-ray diffraction data were collected and crystallographic parameters were determined. The space group was determined to be P3₁21 and the cell parameters to be a=b=86.6 Å and c=205.3 Å. Assuming that three subunits having a molecular weight of 25000 is contained in an asymmetric unit, the solvent content of the crystals is determined to be 58%.

EXAMPLE 4 Crystal Structure Analysis of Wild-Type EB-AP

X-ray diffraction data were collected up to the maximum resolution of 1.9 Å. Since the crystals were severely damaged by the irradiation of X-ray at room temperature, they were rapidly cooled to −173° C., and the measurement was performed. Heavy atom derivatives were screened by immersing the crystals into a solution of heavy metal salts. The diffraction data of heavy atom derivative crystals were obtained by using Rigaku R-AXISIIc. Based on a difference Fourier map with respect to the native data, it was found that K₂PtCl₄ provided good heavy atom isomorphic crystals. The coordinates of the only one platinum binding site of K₂PtCl₄ were obtained by using the program RSPS. These coordinates were further refined by using the program MLPHARE, from which the phase was calculated. By using this phase, five mercury binding sites of the second heavy atom derivative KHgI₄-KI were determined. The heavy atom parameters for both of K₂PtCl₄ and KHgI₄-KI were simultaneously refined by using MLPHARE, and then the phase was improved by performing solvent smoothing and histogram matching by using the program DM. As for K₂PtCl₄, anomalous scattering data were also used. The electron density map calculated by using this good phase was very clear, and thus almost all of amino acid residues were finely fitted.

The first model was constructed on an electron density map prepared with 2.8 Å resolution by using the program QUANTA, and structure refinement was performed by using the program X-PLOR. Electron density was not observed for the six residues at the N-terminus, 135 to 136th residues and one residue at the C-terminus, and thus the structure could not determined uniquely. The final model refined with 1.9 Å resolution (FIG. 4 to FIG. 6) contained 222 residues out of the 231 residues in total, 236 water molecules and one molecule of sulfate ion. The sulfate ion was derived from ammonium sulfate used in the purification process, and was considered to fit to the phosphate binding site of the active center. The crystallographic reliability factor (R factor) determined by using reflections of 8 to 1.9 Å resolution was 21.5%. The average temperature factor was 26 Å² for protein atoms and 45 Å² for water molecules. When a Ramachandran plot was prepared by using the program PROCHECK, it was demonstrated that 93% of residues other than glycine located in the most preferred domain and 7% located at next preferred domain. One subunit was contained in an asymmetric unit and a hexamer was formed by the crystallographic symmetries. The atomic coordinates were represented in FIG. 10 to FIG. 45.

FIG. 4 is a CG photograph representing a crystal structure of the hexamer molecule of EB-AP. The flows of a carbon atoms were represented with ribbon models. Further, the sulfate ion marking the active center was represented with a ball model.

FIG. 5 is a CG photograph representing a crystal structure of a subunit of EB-AP. The flows of a carbon atoms were represented with ribbon models. Further, the sulfate ion marking the active center was represented with a ball model.

FIG. 6 shows the structure of the active site of EB-AP. The sulfate ion is represented at the center. The doted lines represent hydrogen bonds.

FIG. 10 shows the crystallographic data (1) for the structure of EB-AP.

FIG. 11 shows the crystallographic data (2) for the structure of EB-AP.

FIG. 12 shows the crystallographic data (3) for the structure of EB-AP.

FIG. 13 shows the crystallographic data (4) for the structure of EB-AP.

FIG. 14 shows the crystallographic data (5) for the structure of EB-AP.

FIG. 15 shows the crystallographic data (6) for the structure of EB-AP.

FIG. 16 shows the crystallographic data (7) for the structure of EB-AP.

FIG. 17 shows the crystallographic data (8) for the structure of EB-AP.

FIG. 18 shows the crystallographic data (9) for the structure of EB-AP.

FIG. 19 shows the crystallographic data (10) for the structure of EB-AP.

FIG. 20 shows the crystallographic data (11) for the structure of EB-AP.

FIG. 21 shows the crystallographic data (12) for the structure of EB-AP.

FIG. 22 shows the crystallographic data (13) for the structure of EB-AP.

FIG. 23 shows the crystallographic data (14) for the structure of EB-AP.

FIG. 24 shows the crystallographic data (15) for the structure of EB-AP.

FIG. 25 shows the crystallographic data (16) for the structure of EB-AP.

FIG. 26 shows the crystallographic data (17) for the structure of EB-AP.

FIG. 27 shows the crystallographic data (18) for the structure of EB-AP.

FIG. 28 shows the crystallographic data (19) for the structure of EB-AP.

FIG. 29 shows the crystallographic data (20) for the structure of EB-AP.

FIG. 30 shows the crystallographic data (21) for the structure of EB-AP.

FIG. 31 shows the crystallographic data (22) for the structure of EB-AP.

FIG. 32 shows the crystallographic data (23) for the structure of EB-AP.

FIG. 33 shows the crystallographic data (24) for the structure of EB-AP.

FIG. 34 shows the crystallographic data (25) for the structure of EB-AP.

FIG. 35 shows the crystallographic data (26) for the structure of EB-AP.

FIG. 36 shows the crystallographic data (27) for the structure of EB-AP.

FIG. 37 shows the crystallographic data (28) for the structure of EB-AP.

FIG. 38 shows the crystallographic data (29) for the structure of EB-AP.

FIG. 39 shows the crystallographic data (30) for the structure of EB-AP.

FIG. 40 shows the crystallographic data (31) for the structure of EB-AP.

FIG. 41 shows the crystallographic data (32) for the structure of EB-AP.

FIG. 42 shows the crystallographic data (33) for the structure of EB-AP.

FIG. 43 shows the crystallographic data (34) for the structure of EB-AP.

FIG. 44 shows the crystallographic data (35) for the structure of EB-AP.

FIG. 45 shows the crystallographic data (36) for the structure of EB-AP.

EXAMPLE 5 Modeling of the Binding Mode of Wild-Type EB-AP and 5′-Inosinic Acid

Since the Km value of inosine for EB-AP exceeds 100 mM, the affinity is not high enough to determine the binding mode by X-ray crystallography. In fact, when compounds serving as an inhibitor of EB-AP such as glucose-6-sulfate and adenosine-thiomonophosphate were soaked into the crystals of wild-type EB-AP and X-ray diffraction data were collected to prepare an electron density map, electron density corresponding to these compounds was not observed. Therefore, it was decided to predict the binding style of 5′-inosinic acid and EB-AP by using computer graphics (so-called docking study). As the program, QUANTA was used. Since a sulfate ion was found at the center of the active site in the crystal structure, the phosphate group of 5′-inosinic acid was superimposed on it. Further, since it was known that mutations of G74D and I153T would reduce the Km value of 5′-inosinic acid for EB-AP, it was judged that 5′-inosinic acid would bind to a position not far from G74 and I153, and decided the position of 5′-inosinic acid. At that time, the position was decided so that the atoms constituting 5′-inosinic acid and the atoms constituting EB-AP should not interfere with each other. In the model constructed as described above, if I153 is changed to Thr, the γ-oxygen atom of the side chain of the threonine introduced by the replacement and 2′-hydroxyl group of the ribose of inosine form a hydrogen bond. Further, when the electrostatic potential of EB-AP was displayed by using the program GRASP, the positively charged inosine base interacted with a negatively charged domain of the surface of the EB-AP molecule, which suggested that a model was reasonable.

EXAMPLE 6 Crystal Structure Analysis of G74D/I153T Mutant EB-AP

The G74D/I153T mutant EB-AP has an increased ratio of the transphosphorylation activity relative to the phosphatase activity, and in connection with this fact, the nucleoside-5′-phosphate producing ability is also improved. It is considered that this is caused by the decrease of the Km value for a nucleoside, i.e., improvement of the affinity for a nucleoside. It was expected that the molecular mechanism for improvement of the affinity for a nucleoside should be elucidated by determining the crystal structure of the mutant EB-AP and comparing it with the crystal structure of wild-type EB-AP.

At room temperature, the X-ray diffraction data were collected to the maximum resolution of 2.4 Å. Estimating from the volume of the unit cell, space group and molecular weight of the enzyme, it was expected that one hexamer molecule is contained in the asymmetric unit. Then, as a model for searching the hexamer structure of wild-type EB-AP, analysis was performed by the molecule replacement method using the program amore. Data of 10-3 Å resolution were used for the rotation search, and data of 10-4 Å resolution were used for the translation search. In the both searches, the correct answer appeared as a top peak. When refinement was performed by considering the molecule as a rigid body, the R factor decreased to 37.3%. Then, structure modification on graphics using QUANTA and structure refinement using X-PLOR were repeated to obtain a model with an R factor of 19.9% at 10-2.4 Å resolution.

When a docking model of 5′-inosinic acid and the G74D/I153T mutant EB-AP was prepared in the same manner as in Example 5, it was expected that the y-oxygen atom of the side chain of the Thr153 introduced by the replacement would form a hydrogen bond with a hydroxyl group of ribose of inosine. By comparing temperature factors, it was found that the fluctuation of the loop containing Asp74 where another replacement was made was larger in the G74D/I153T mutant EB-AP compared with the wild-type. This loop is expected to interact with the base of inosine, and it is suggested that it becomes more likely to bind to the base due to the larger fluctuation.

EXAMPLE 7 Crystal Structure Analysis of the Complex of Wild-Type EB-AP with Molybdate (Reaction Intermediate Analogue)

In the enzymatic reaction of EB-AP, a phosphoric acid monoester linkage is first cleaved, and the phosphate group forms a covalent bond with an active site residue, His189. The enzyme molecule in this state is called a reaction intermediate. The reaction intermediate is quickly attacked by water or alcohol, and as a result, a phosphate ion dissociates from it. When water attacks, phosphatase activity will be exerted, and when alcohol attacks, the transphosphorylation activity will be exerted. In the both cases, the reaction intermediate is unstable and it is impossible to determine its structure by X-ray crystallography. However, a complex (reaction intermediate analogue) in which molybdate covalently bonds to His189 instead of phosphate is not attacked by water, and thus it exists stably.

In the transphosphorylation reaction, a phosphate acceptor binds to the reaction intermediate to form a phosphoric acid monoester linkage. Therefore, for the purpose of modeling the binding mode with a nucleoside, it is more suitable to use a reaction intermediate structure rather than the structure in free form. In order to perform docking study of the reaction intermediate and a nucleoside, the crystal structure of the reaction intermediate analogue was determined.

At room temperature, the X-ray diffraction data were collected to the maximum resolution of 2.4 Å. Estimating from the volume of unit cell, space group and molecular weight of the enzyme, it was expected that a half of the hexamer, i.e., three subunits, were contained in the asymmetric unit. Therefore, trimer structure in which the subunits were correlated with one another with a three-fold axis was prepared as a model for searching in the molecule replacement method. Data of 10-3 Å resolution were used for the rotation search, and data of 10-4 Å resolution were used for the translation search. In the both searches, the correct answer appeared as a top peak. When refinement was performed by considering the molecule as a rigid body, the R factor decreased to 42.4%. Then, structure modification on graphics using QUANTA and structure refinement using X-PLOR were repeated to obtain a model showing an R factor of 22.3% at 8-2.4 Å resolution. The asymmetric unit contained a half of the hexamer, i.e., three subunits.

EXAMPLE 8 Crystallization and Crystal Structure Analysis of G72D/I151T Mutant Enzyme Derived From Acid Phosphatase of Morganella morganii (MM-AP)

Crystallization of G72D/I151T double mutation mutant of MM-AP was performed by using vapor diffusion in the hanging drop method. A solution of the protein (concentration: 40 mg/ml) and a 125 mM citrate buffer (pH 4.8) containing 25% (w/v) of polyethylene glycol 1000, 25 mM ammonium sulfate and 25 mM DTT in the same amounts (5 μl each) were dropped and mixed on siliconized cover glass, and a well filled with 500 p1 of 125 mM citrate buffer (pH 4.8) containing 25% (w/v) of polyethylene glycol 1000, 25 mM ammonium sulfate and 25 mM DTT was covered with the cover glass so that a drop of the mixed solution should be hung above the well and left stand at 20° C. Crystals appeared within a few days, and they grew up to a measurable size (about 0.4×0.4×0.3 mm) within 1 week.

Using an X-ray diffraction apparatus, R-AXIS IIc of Rigaku Co., Ltd., X-ray diffraction data were collected to determine crystallographic parameters. The space group was determined to be P2₁2₁2₁ and the cell parameters to be a=90.64 Å, b=119.74 Å and c=136.14 Å. The diffraction data to 2.6 Å resolution data were measured at 100 K on the synchrotron radiation light facility BL-6B at the HIGH ENERGY ACCELERATOR RESEARCH ORGANIZATION, Tsukuba.

Estimating from the volume of unit cell, space group and molecular weight of the enzyme, it was expected that one hexamer molecule was contained in the asymmetric unit. Then, by using the hexamer structure of wild-type EB-AP as a model for searching, the molecule replacement method was performed using the program amore. Data of 10-3 Å resolution were used for the rotation search, and data of 10-4 Å resolution were used for the translation search. In the both searches, the correct answer appeared as a top peak. After refinement was performed by considering the molecule as a rigid body, structure modification on graphics using QUANTA and structure refinement using X-PLOR were repeated to obtain a model with an R factor of 0.197% at 10-2.6 Å resolution.

The distances between Ca atoms of the five active residues shown in FIG. 1 (Lys113, Arg120, His148, Arg181 and His187) are shown in Table 2. It was confirmed that, in MM-AP that is an analogous enzyme of EB-MP, all of the interatomic distances between the active residues fell within the ranges defined based on the three-dimensional structure of EB-AP.

TABLE 2 G72D/I151T mutant MM- Lower Upper AP limit limit Lys113 Arg120 11.3Å 10.4Å 12.6Å His148 12.6Å 11.3Å 13.8Å Arg181 16.3Å 14.5Å 17.4Å His187 12.5Å 10.7Å 13.6Å Arg120 His148 14.0Å 12.2Å 15.2Å Arg181 10.9Å  9.4Å 11.8Å His187  6.1Å  4.5Å  6.7Å His148 Arg181  8.9Å  6.7Å  9.8Å His187 10.2Å  8.8Å 11.0Å Arg181 His187  5.4Å  4.5Å  6.8Å

EXAMPLE 9 Modeling of the Binding Mode of EB-AP Reaction Intermediate and Inosine

A binding mode model was constructed on computer graphics by using QUANTA (FIG. 3). Molybdate was replaced with phosphate as it was. Inosine was placed near the nucleoside portion of 5′-inosinic acid in the binding mode model of wild-type EB-AP and 5′-inosinic acid. However, the degree of freedom was of course higher than the docking with 5′-inosinic acid, since an inosine did not have a phosphoric acid monoester linkage. Therefore, fine adjustment of the position of inosine was performed so that inosine should bind to the molecular surface of EB-AP in a more preferred condition to obtain a binding style model. For the subsequent designing of a mutant enzyme, this model was used.

EXAMPLE 10 Design of Mutant EB-AP Aiming at Enhancement of Affinity for Nucleoside

According to the model constructed in Example 8, it was suggested that the side chain of Ser72 might interact with the base of inosine. It was expected that, if this residue was replaced with an aromatic amino acid such as phenylalanine, tyrosine and tryptophan, the π—π interaction would be generated between an aromatic ring and a nucleoside base and thus the affinity of nucleoside for EB-AP would be improved. Similarly, it was expected that, if it was replaced with an amino acid having a branched hydrophobic group in the side chain such as valine, leucine and isoleucine, the CH/π interaction would be generated between the branched hydrophobic group in the side chain and the nucleoside base, or if it was replaced with a negatively charged amino acid such as glutamic acid and an aspartic acid, it would be electrostatically attracted by the positive charge of the nucleoside base, and thus the affinity would be improved. Therefore, in order to further enhance the transphosphorylation activity of the G74D/I153T mutant EB-AP of which transphosphorylation activity was relatively increased compared with the phosphatase activity, S72F, S72Y, S72W, S72V, S72E and S72D mutants of this mutant EB-AP were prepared. In addition, mutants in which S72 was replaced with the other amino acids were also produced. Incidentally, these mutants would become a 3-residue replaced mutant EB-AP.

EXAMPLE 11 Construction of 3-Residue Replaced Mutant of EB-AP in which Ser72 is Replaced with Another Amino Acid

In order to construct a mutant EB-AP for expression in Escherichia coli JM109, a plasmid pEPI340 containing a gene for G74D/I153T mutant EB-AP was used as a template for site-directed mutagenesis utilizing PCR. The nucleotide sequences of these plasmids pEPI305 and pEPI340 are disclosed in Japanese Patent Laid-open (Kokai) No. 10-201481/1998, paragraph (0143), Table 12. A strain of Escherichia coli JM109 harboring the plasmid pEPI305 was designated as AJ13144, and deposited as an international deposition at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 1–3, Higashi, 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Feb. 23, 1996, and it received an accession number of FERM BP-5423 [refer to the description of the aforementioned patent document, paragraphs (0105) to (0110)].

The mutation was introduced by using Quick Change site-directed mutagenesis kit available from Stratagene, United States, and primers corresponding to each mutant enzyme (FIG. 7, SEQ ID NOS: 11–61) according to the manufacturer's protocol. Escherichia coli XL-1 was transformed with the PCR product. The transformed cells were plated on an L agar medium plate containing 100 μl/ml of ampicillin, and incubated at 37° C. for 16 hours. The produced colonies were collected, and cultured overnight in L medium containing 100 μl/ml of ampicillin with shaking. The cells were collected from the medium by centrifugation, and the plasmid was extracted by using FlexiPrep Kit available from Pharmacia (Sweden) according to the manufacturer's protocol. The nucleotide sequences coding for the various 3-residue replaced mutant EB-AP were confirmed by DNA sequence analysis.

The synthesis of the primers shown in FIG. 7 was consigned to Japan BioService Co., Ltd.

EXAMPLE 12 Measurement of Transphosphorylation Activity and Reaction Rate Constants of Mutant EB-AP

Escherichia coli JM109 transformed with a plasmid containing a gene for one of various 3-residue replaced mutant EB-AP's was inoculated into 50 ml of L medium containing 100 μl/ml of ampicillin and cultured at 37° C. for 16 hours. The cells were collected by centrifugation from the medium, suspended in 3 ml of 25 mM phosphate buffer (pH 7.0), and disrupted by sonication at 4° C. for 20 minutes. The insoluble fraction in the sonicated suspension was removed by centrifugation to prepare a cell free extract. The expression of each EB-AP 3-residue replaced mutant enzyme was confirmed by SDS-PAGE. The expression amount was about 20% of the total protein.

The transphosphorylation activity of the cell free extract was measured under the following conditions. A reaction mixture (1 ml) containing 2 mM inosine, 100 mM sodium pyrophosphate, 100 mM acetate buffer (pH 4.0) and 100 μl of the cell free extract was incubated at pH 4 and 30° C. for 10 minutes. After 200 μl of 1 N hydrochloric acid was added to stop the reaction, the precipitates were removed by centrifugation, and the produced 5′-inosinic acid was quantified. The transphosphorylation activity of each 3-residue replaced mutant EB-AP was represented with a relative activity, which was a ratio of the amount of 5′-inosinic acid produced by the 3-residue replaced mutant over that produce by the G74D/I153T mutant, to which the third mutation was introduced.

Then, the Km value for inosine in the transphosphorylation reaction by each 3-residue replaced mutant EB-AP was measured under the following conditions. A reaction mixture (1 ml) containing 100 mM sodium pyrophosphate, 100 mM acetate buffer (pH 4.0), 10–100 mM inosine and 100 μl of the cell free extract was incubated at pH 4 and 30° C. for 10 minutes. After 200 μl of 1 N hydrochloric acid was added to stop the reaction, the precipitates were removed by centrifugation, and the produced 5′-inosinic acid was quantified. The Km value was calculated by the Hanes-Woolf plotting. The results are shown in Table 3.

TABLE 3 Transphosphorylation Km value activity S72F/G74D/I153T 20 mM 2.80 S72Y/G74D/I153T 25 mM 2.04 S72W/G74D/I153T 30 mM 1.71 S72D/G74D/I153T 33 mM 1.59 S72V/G74D/I153T 40 mM 2.46 S72E/G74D/I153T 40 mM 3.19 S72M/G74D/I153T 46 mM 1.94 S72T/G74D/I153T 50 mM 1.91 S72L/G74D/I153T 57 mM 2.24 S72R/G74D/I153T 59 mM 1.99 S72Q/G74D/I153T 77 mM 2.42 S72K/G74D/I153T 78 mM 1.53 S72P/G74D/I153T 109 mM 1.34 S72A/G74D/I153T 115 mM 0.78 S72N/G74D/I153T 124 mM 0.43 S72G/G74D/I153T 137 mM 0.43 S72H/G74D/I153T N.D. N.D. G74D/I153T 100 mM 1.00 10-Residue replaced 40 mM 1.44 mutant enzyme

In Example 10, all of the mutants expected to have improved affinity for inosine due to the π—π interaction, the CH/π interaction and the electrostatic interaction (S72F, S72Y, S72W, S72V, S72E, S72D) showed decreased Km value for inosine compared with that of the G74D/I153T mutant EB-AP, in which Ser72 is unchanged, and thus they showed improved affinity for inosine. Further, they also showed improvement in transphosphorylation activity. In particular, the S72F mutant showed marked improvements in both of the Km value and the transphosphorylation activity. It is estimated that the aromatic ring of phenylalanine and the inosine base caused the π—π interaction in suitable positional relationship, and thus the affinity was improved. The Km values of S72M, S72T, S72R, S72Q and S72K mutants also decreased. It was considered that some preferred interactions such as hydrophobic interaction and formation of hydrogen bonds were caused between these amino acid residues and the nucleoside base. Incidentally, a gene could not be produced for S72I. Further, considering the possibility that the S72C mutation causes an erroneous S—S bond, it was not produced.

5′-Inosinic acid was analyzed by high performance liquid chromatography (HPLC) under the following conditions.

-   Column: Cosmosil 5C18-AR (4.6×150 mm), produced by Nakarai Tesque -   Mobile phase: 5 mM potassium phosphate buffer (pH 2.8)/methanol=95/5 -   Flow rate: 1.0 ml/min -   Temperature: room temperature -   Detection: UV 245 nm

Example 13 Production of 5′-Inosinic Acid Using Escherichia coli JM109 to which S72F/G74D/I153T Mutant EB-AP Gene is Introduced

Escherichia coli JM109 transformed with a plasmid containing a gene for G74D/I153T mutant, 10-residue replaced mutant or S72F/G74D/I153T mutant EB-AP was inoculated into 50 ml of L medium containing 100 μg/ml of ampicillin and 1 mM of IPTG, and cultured at 37° C. for 16 hours.

Cells of Escherichia coli JM109 introduced with each of the aforementioned mutant EB-AP genes were added at a density of 100 mg/dl in terms of dry cell weight to a solution in an acetate buffer (pH 4.0) containing 12 g/dl of pyrophosphoric acid and 6 g/dl of inosine, and reaction was performed at 30° C. for 24 hours while pH was maintained at 4.0. The results of measurement of the produced amount of 5′-inosinic acid are shown in Table 4. The produced inosinic acid consisted only of 5′-inosinic acid, and 2′-inosinic acid and 3′-inosinic acid byproducts were not observed at all.

In the reaction utilizing Escherichia coli JM109 introduced with the plasmid containing the G74D/I153T mutant EB-AP gene, 7.5 g/dl of 5′-inosinic acid was produced and accumulated, but the accumulation did not increase even if the reaction time was prolonged. In the reaction utilizing Escherichia coli JM109 introduced with the plasmid containing the 10-residue replaced mutant EB-AP gene, the accumulation was improved, and 12.1 g/dl of 5′-inosinic acid was produced and accumulated. In the reaction utilizing Escherichia coli JM109 transformed with the plasmid containing the S72F/G74D/I153T mutant EB-AP gene, which was designed and constructed based on the three-dimensional structure, the productivity was further improved, and 13.2 g/dl of 5′-inosinic acid was produced and accumulated.

TABLE 4 Produced inosinic acid Introduced mutant enzyme gene (g/dl) G74D/I153T 7.5 10-Residue replaced mutant 12.1 S72F/G74D/I153T 13.2

EXAMPLE 14 Measurement of Transphosphorylation Activity and Reaction Rate Constant of 3-Residue Replaced Mutant EB-AP, in which L16W, S71W, S73W, E104F or E104W Mutation is Introduced

Since it was considered that the S72F mutation improved the affinity for inosine by the π—π interaction, other amino acid residues that could be expected to cause the π—π interaction with the inosine base by replacement with an aromatic amino acid were searched on computer graphics. As a result, there was suggested possibility that an aromatic ring introduced by L16W, S71W, S73W, E104F and E104W might interact with the inosine base. Therefore, these five kinds of 3-residue replaced mutant EB-AP's were prepared (based on the G74D/I153T mutant EB-AP) by the method described in Example 11 (primers corresponding to each mutant enzyme were shown in FIG. 8A, SEQ ID NOS: 62–76), and their transphosphorylation activity and reaction rate constant were measured by the method described in Example 12. The results are shown in Table 5. Although the transphosphorylation activity decreased in all of the mutant enzymes, the Km value decreased in all of the mutant enzymes and thus it was suggested that the affinity for inosine was improved. Although Leu16 was separated by 10 Å (in terms of the distance between Cα's) from Ser72, which was considered to surely interacts with inosine, it was demonstrated that the interaction with inosine was possible, even if it was separated by such a distance.

The synthesis of the primer sets shown in FIG. 8A was consigned to Japan BioService Co., Ltd.

TABLE 5 Transphosphorylation Km value activity L16W/G74D/I153T 33 mM 0.21 S71W/G74D/I153T 75 mM 0.26 S73W/G74D/I153T 29 mM 0.77 E104F/G74D/I153T 61 mM 0.65 E104W/G74D/I153T 67 mM 0.26 G74D/I153T 100 mM 1.00 10-Residue replaced 40 mM 1.44 mutant enzyme

EXAMPLE 15 Construction of 10-Residue Replaced Mutant EB-AP, in which A72F or A72E Mutation is Introduced, and Measurement of Transphosphorylation Activity and Reaction Rate Constant Thereof

The S72F mutation that decreased the Km value in the highest degree and the S72E mutation that enhanced the activity in the highest degree in Example 12 were introduced into the 10-residue replaced mutant EB-AP. Since Ser72 was replaced with Ala in the 10-residue replaced mutant EB-AP, the A72F and A72E mutations were actually introduced. When a wild-type EB-AP is considered as standard, ten residues were replaced in the both. These two kinds of mutants were produced by the method described in Example 11 (primers corresponding to each mutant enzyme are shown in FIG. 8B, SEQ ID NOS: 77–82). As a template for site-directed mutagenesis utilizing PCR, plasmid pEMP370 (Japanese Patent Laid-open (Kokai) No. 9-37785/1997, Example 19) containing a 10-residue replaced mutant EB-AP gene was used. Furthermore, transphosphorylation activity and reaction rate constant were measured by the method described in Example 12. The results are shown in Table 6. The transphosphorylation activity was represented in terms of relative activity, which was a ratio of the amount of 5′-inosinic acid produced by the 3-residue replaced mutant over that produce by the G74D/I153T mutant, to which the third mutation was introduced. In both of the mutant enzymes, the Km value markedly decreased. As for the transphosphorylation activity, it was decreased by the A72F mutation, whereas it was increased by A72E mutation.

TABLE 6 Transphosphorylation Km value activity A72F/10-residue replaced 9 mM 0.11 mutant enzyme A72E/10-residue replaced 15 mM 2.30 mutant enzyme 10-residue replaced mutant 40 mM 1.44 enzyme

EXAMPLE 16 Production of 5′-Inosinic Acid Utilizing Escherichia coli JM109 to which Gene for A72F/10-Residue Replaced Mutant EB-AP or Gene for A72E/10-Residue Replaced Mutant EB-AP is Introduced

Experimental production of 5′-inosinic acid was performed by the method described in Example 13 using Escherichia coli JM109 transformed with a plasmid containing the genes for A72E/10-residue replaced mutant or A72F/10-residue replaced mutant EB-AP. The results are shown in Table 7. The accumulation amount of 5′-inosinic acid increased in the both mutants.

TABLE 7 Introduced mutant enzyme Produced inosinic acid gene (g/dl) A72F/10-residue replaced 13.9 mutant A72E/10-residue replaced 13.9 mutant 10-residue replaced mutant 12.1

EXAMPLE 17 Construction of Mutant EB-AP, in which I103D and T153N Mutations are Introduced, and Measurement of Transphosphorylation Activity and Reaction Rate Constant

The model shown in FIG. 3 suggested that Asp introduced by the I103D mutation caused electrostatic interaction with the inosine base and Asn introduced by the T153N mutation formed a hydrogen bond with a hydroxyl group of ribose. Therefore, I103D/G74D/I153T mutant EB-AP and G74D/I153N mutant EB-AP were produced by introducing these residues into the G74D/I153T mutant EB-AP in a manner described in Example 11 (primers corresponding to each mutant enzyme were shown in FIG. 8C, SEQ ID NOS: 83–88). Further, transphosphorylation activity and reaction rate constant were measured by the method described in Example 12. The results are shown in Table 8. The transphosphorylation activity was represented in terms of relative activity, which was a ratio of the amount of 5′-inosinic acid produced by the 3-residue replaced mutant over that produce by the G74D/I153T mutant, to which the third mutation was introduced. In the both mutants, the transphosphorylation activity decreased, whereas the Km value decreased. Thus, it was demonstrated that the affinity for inosine was improved.

TABLE 8 Transphosphorylation Km value activity I103D/G74D/I153T 51 mM 0.09 G74D/I153N 38 mM 0.18 G74D/I153T 100 mM 1.00

EXAMPLE 18 Construction of Mutant EB-AP in which Leu140 is Replaced with Phe, Glu or Lys and Measurement of Transphosphorylation Activity and Reaction Rate Constant

Although Leu140 is separated from Ser72 more than 10 Å, it is located at a position immediately adjacent to the phosphate binding site in the three-dimensional structure of the reaction intermediate analogue. Therefore, it was considered that, if this residue was replaced, the structure around the phosphate binding site of the reaction intermediate would be changed, and as a result, the structure and fluctuation of the nucleoside binding site would also be affected. It is expected that, if this residue is replaced with more bulky Phe, positively charged Lys or negatively charged Glu, the affinity for a nucleoside may be changed. It was decided that the mutations should be introduced into the A72E/10-residue replaced enzyme, which showed high transphosphorylation activity in Example 15. These three kinds of mutants were produced by the method described in Example 11 (primers corresponding to each mutant enzyme are shown in FIG. 8D, SEQ ID NOS: 89–97). As a template for site-directed mutagenesis utilizing PCR, a plasmid containing the gene for A72E/10-residue replaced mutant EB-AP was used. Furthermore, transphosphorylation activity and reaction rate constant were measured by the method described in Example 12. The results are shown in Table 9. The transphosphorylation activity was represented in terms of relative activity, which was a ratio of the amount of 5′-inosinic acid produced by the 3-residue replaced mutant over that produce by the G74D/I153T mutant, to which the third mutation was introduced.

TABLE 9 Transphosphorylation Km value activity A72E/L140F/10-residue 9 mM 1.66 replaced mutant enzyme A72E/L140K/10-residue 78 mM 0.07 replaced mutant enzyme A72E/L140E/10-residue 322 mM 0.16 replaced mutant enzyme A72E/10-residue replaced 15 mM 2.30 mutant enzyme

The mutant, in which L140F mutation is introduced, showed decreased Km value. Conversely, L140K and L140E mutations markedly increased Km.

EXAMPLE 19 Purification of Wild-Type Acid Phosphatase Derived from Enterobacter Aerogenes IFO12010 and Determination of N-Terminus Amino Acid Sequence Thereof

An acid phosphatase derived from Enterobacter aerogenes IFO12010 was purified from cultured cells of Escherichia coli JM109/pENP110 described in Japanese Patent Laid-open (Kokai) No. 10-201481/1998, Example 24, and its N-terminus amino acid sequence was determined to determine the amino acid sequence of the mature protein. Escherichia coli JM109/pENP110 is a bacterium prepared by introducing an acid phosphatase gene derived from Enterobacter aerogenes IFO12010 into Escherichia coli JM109 strain, and it produces the acid phosphatase. The amino acid sequence of a precursor protein deduced from the nucleotide sequence of this acid phosphatase gene corresponds to the sequence shown in SEQ ID NO: 10. The amino acid sequence shown in SEQ ID NO: 10 is an amino acid sequence of L61Q/A63Q/E64A/N67D/S69A/G72D/T133K/E134D/I151T mutant EA-AP.

50 ml of the nutrient medium (pH 7.0) containing 1 g/dl of peptone, 0.5 g/dl of yeast extract and 1 g/dl of sodium chloride was put into 500-ml Sakaguchi flask, and sterilized by heating at 120° C. for 20 minutes. One platinum loop of Escherichia coli JM109/pENP110 was inoculated into the medium, and cultured at 30° C. for 16 hours with shaking. The cells were collected from the medium by centrifugation, suspended in 100 mL of 100 mM potassium phosphate buffer (pH 7.0), and disrupted by sonication at 4° C. for 20 minutes. The insoluble fraction in the sonicated suspension was removed by centrifugation to prepare a cell free extract. Ammonium sulfate was added to this cell free extract to 30% saturation. After the produced precipitates were removed by centrifugation, ammonium sulfate was further added to the supernatant solution to 60% saturation. The produced precipitates were collected by centrifugation and dissolved in 100 mM potassium phosphate buffer. This crude enzyme solution was dialyzed three times against 500 mL of 100 mM potassium phosphate buffer (pH 7.0), then loaded on a DEAE-Toyopearl 650M column (φ3.0×10.0 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.0), and washed with 20 mM potassium phosphate buffer (pH 7.0). Since the transphosphorylation activity was found in a passing fraction, that fraction was collected. The fraction was added with ammonium sulfate to 35% saturation, and adsorbed on a Butyl Toyopearl column (+3.0×7.0 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.0) containing 35% saturated ammonium sulfate. The fraction was eluted with a linear concentration gradient of 35% saturated to 20% saturated potassium phosphate buffer (pH 7.0). The active fractions were collected, dialyzed against 1 L of 10 mM potassium phosphate buffer (pH 6.0), and adsorbed on a CM-Toyopearl column (φ3.0×7.0 cm) equilibrated with 10 mM potassium phosphate buffer (pH 6.0). The fractions were eluted with a linear concentration gradient of potassium phosphate buffer (pH 6.0) containing 0 mM to 300 mM potassium chloride. The active fractions were collected.

As a result of the above procedure, an enzyme exhibiting transphosphorylation activity was finally purified by about five times from the cell free extract at a recovery ratio of about 16%. This enzyme preparation was confirmed to be uniform by SDS-polyacrylamide gel electrophoresis.

This purified enzyme was adsorbed on a DITC membrane (produced by Milligen/Biosearch), and the amino acid sequence of its N-terminus was determined by using Prosequencer 6625 (produced by Milligen/Biosearch). As a result, the amino acid sequence of the five residues at the N-terminus was determined, which is shown in SEQ ID NO: 98. Since the N-terminus of the purified enzyme was started from the 21st leucine residue (amino acid number 1) of the sequence of SEQ ID NO: 10, it was considered that the amino acid sequence shown in SEQ ID NO: 10 was a sequence of a precursor protein, and the peptide from the 1st methionine residue (amino acid number 20) to the 20th alanine residue (amino acid number 1) was removed after translation. Based on this result, it was considered that the amino acid sequence of the mature protein corresponded to the sequence of the amino acid numbers 1–228 of the sequence shown in SEQ ID NO: 10.

EXAMPLE 20 Construction of Gene for Mutant Enzyme of Acid Phosphatase Derived from Enterobacter aerogenes (EA-AP) and Production of 5′-Inosinic Acid Using Escherichia coli JM109 to which that Gene is Introduced

It was decided that a mutation homologous to the three mutations of S72F/G74D/I153T, which enhanced the transphosphorylation activity for inosine in EB-AP, was introduced into EA-AP. The result of alignment of the amino acid sequences of EB-AP and EA-AP (wild-type) performed by using the program BLAST is shown in FIG. 9. It was demonstrated that Ser72/Gly74/Ile153 of EB-AP corresponded to Ala70/Gly72/Ile151 in EA-AP. Therefore, A70F/G72D/I151T mutant EA-AP was produced by the method described in Example 11. 5′-Inosinic acid was produced from inosine by using Escherichia coli JM109 transformed with a plasmid containing the mutant enzyme gene by the method described in Example 13. The results are shown in Table 10. The A70F/G72D/I151T mutant EA-AP showed 5′-inosinic acid producing ability comparable to that of the S72F/G74D/I153T mutant EB-AP.

TABLE 10 Introduced mutant enzyme Produced inosinic acid gene (g/dl) EA-AP A72F/G74D/I153T 13.1 EB-AP S72F/G74D/I153T 13.2

FIG. 9 shows the result of amino acid sequence alignment of EB-AP and the acid phosphatase derived from Enterobacter aerogenes (EA-AP) performed by using the program BLAST. The upper row indicates EB-AP and the lower row indicates EA-AP. In the middle row, if the residues of the both are identical, the name of the residue is indicated, and if they are analogous even though they are not identical, + is indicated. The position of the 72nd residue (Ser72) of EB-AP was indicated with [72]. The corresponding residue in EA-AP is Ala70.

EXAMPLE 21 High Expression of Enzyme Caused by Modification of Promoter Sequence in Novel Mutant Acid Phosphatase Gene Derived from Enterobacter Aeroagenes IFO12010

A site-specific mutation was introduced by a genetic engineering technique into the promoter sequence segment of the gene coding for the mutant acid phosphatase derived from Enterobacter aerogenes IFO12010 to construct a gene coding for a mutant acid phosphatase exhibiting increased enzyme expression amount. As a gene for introducing a mutation, a plasmid pENP170 that contained a mutant EA-AP gene coding for L61Q/A63Q/E64A/N67D/S69A/G72D/T133K/E134D/I151T mutant EA-AP was used. pENP170 was prepared as follows.

From a plasmid pENP110 containing the gene coding for a wild-type acid phosphatase derived from Enterobacter aerogenes IFO12010 obtained by the method described in Japanese Patent Laid-open (Kokai) No. 10-201481/1998, Example 24, a DNA fragment having a size of 1.6 kbp and containing a gene coding for the wild-type acid phosphatase was excised with restriction enzymes SalI and KpnI, and ligated to pUC19 (product produced by Takara Shuzo Co. Ltd.) digested with SalI and KpnI. This plasmid was designated as pENP120. The following mutations were introduced into pENP120 by site-specific mutagenesis to obtain pENP170. The nucleotide sequence of the SalI-KpnI 1.6 kbp DNA fragment in pENP170 was a sequence shown in SEQ ID NO: 9.

-   72Gly (GGC)->Asp (G*AC) -   151Ile (ATC)->Thr (A*CC) -   61Leu (CTG)->Gln (C*AG) -   63Ala (GCT)->Gln (*C*A*G) -   64Glu (GAA)->Ala (G*CA) -   67Asn (AAC)->Asp (*GAC) -   69Ser (AGC)->Ala (*G*CC) -   133Thr (ACC)->Lys (A*A*A) -   134Glu (GAG)->Asp (GA*C)

The mutations were introduced into the promoter sequence segment of the mutant EA-AP gene contained in pENP170 by using Quick Change site-directed mutagenesis kit produced by Stratagene. The mutation was introduced according to the protocol of Stratagene by using oligonucleotides for introduction of mutation, MUT170 (SEQ ID NO: 99) and MUT171 (SEQ ID NO: 100), which were synthesized by using a DNA synthesizer (Model 394 produced by Applied Biosystem), and pENP170 as a template.

Escherichia coli JM109 (produced by Takara Shuzo) was transformed with the obtained plasmid DNA in a conventional manner. The cells were plated on L agar medium containing 100 μg/ml of ampicillin to obtain transformants. A plasmid was prepared from the transformants by the alkali lysis method, and their nucleotide sequence was determined to confirm that the target nucleotides were replaced. The determination of the nucleotide sequences was performed according to the method of Sanger et al. (J. Mol. Biol., 143, 161 (1980)) using Taq DyeDeoxy Terminator Cycle Sequencing Kit (produced by Applied Biochemical).

As described above, a mutant gene was constructed, in which the nucleotide sequence of −10 region of the deduced promoter sequence located upstream from the coding region of the deduced acid phosphatase derived from Enterobacter aerogenes IFO12010 was changed from AAAAAT to TATAAT, which was the same as lac promoter of Escherichia coli. The plasmid containing this mutant gene was designated as pENP180.

Escherichia coli JM109/pENP170 and Escherichia coli JM109/pENP180 introduced with the gene of which −10 region of the promoter sequence was modified were each inoculated to 50 ml of L medium containing 100 μg/ml of ampicillin and 50 ml of L medium containing 100 μg/ml of ampicillin added with 1 mM of IPTG, respectively, and cultured at 37° C. for 16 hours. The cells were collected by centrifugation from each medium of each strain, and washed once with physiological saline. The cells of each culture was added in an amount of 100 mg/dl in terms of dry cell weight to a solution containing 15 g/dl of pyrophosphoric acid and 8 g/dl of inosine dissolved in 100 mM acetate buffer (pH 4.0), and allowed to react at 30° C. for 1 hour while pH was maintained at 4.0. The amounts of the produced 5′-inosinic acid are shown in Table 11.

Inosine and 5′-inosinic acid were analyzed by high performance liquid chromatography (HPLC) under the following conditions.

-   Column: Cosmosil 5C18-AR (4.6×150 mm, produced by Nakarai Tesque) -   Mobile phase: 5 mM potassium phosphate buffer (pH 2.8)/methanol=95/5 -   Flow rate: 1.0 ml/min -   Temperature: room temperature -   Detection: UV 245 nm

Escherichia coli JM109/pENP170 showed low activity with no addition of IPTG, whereas Escherichia coli JM109/pENP180 showed high activity even with no addition of IPTG. Further, Escherichia coli JM109/pENP180 showed further higher activity with addition of IPTG. Thus, the effectiveness of the modification of the promoter region was demonstrated.

TABLE 11 Produced 5′-inosinic Strain IPTG acid (g/dl) Escherichia coli Not added 0.73 JM109/pENP170 Addition of 1 mM 3.09 Escherichia coli Not added 2.86 JM109/PENP180 Addition of 1 mM 3.37

EXAMPLE 22 Construction of Gene Coding for Novel Mutant Acid Phosphatase Derived from Enterobacter Aerogenes IFO01210 Showing Improved Affinity for Nucleoside

A site specific mutation was introduced into the mutant acid phosphatase gene derived form Enterobacter aerogenes IFO12010 constructed in Example 21 by a genetic engineering technique to prepare a gene coding for a mutant acid phosphatase of which affinity for a nucleoside, in particular, guanosine, was improved. As the replacements of amino acid residues, a combination of the amino acid residue replacements identified to contribute to the enhancement of the affinity based on the three-dimensional structure analysis of the Escherichia blattae enzyme was introduced.

The mutations were introduced into the plasmid DNA by using Quick Change site-directed mutagenesis kit produced by Stratagene. Twenty kinds of oligonucleotides (Table 12) for introduction of mutation from MUT180 (SEQ ID NO: 101) to MUT521 (SEQ ID NO: 120) were synthesized by using a DNA synthesizer (Model 394 produced by Applied Biosystem). The mutations were introduced according to the protocol of Stratagene by using pENP170 as the first template and MUT180 and MUT181 as oligonucleotides for introduction of mutation.

Escherichia coli JM109 (produced by Takara Shuzo) was transformed with each obtained plasmid DNA in a conventional manner. The cells were plated on L agar medium containing 100 μg/ml of ampicillin to obtain transformants. A plasmid was prepared from the transformants by the alkali lysis method, and the nucleotide sequence was determined to confirm that the target nucleotide was replaced. The determination of the nucleotide sequence was performed according to the method of Sanger et al. (J. Mol. Biol., 143, 161 (1980)) using Taq DyeDeoxy Terminator Cycle Sequencing Kit (produced by Applied Biochemical). As described above, a gene coding for a mutant acid phosphatase was constructed, in which 153rd threonine residue (ACC) was replaced with a serine residue (TCC), and a plasmid containing this mutant gene was designated as pENP200.

By using a plasmid introduced with a mutation as a new template, the same procedure was repeated to cumulatively introduce site-specific mutations. Plasmids were prepared from the transformants by the alkali lysis method, and their nucleotide sequences were determined to confirm that the target nucleotides were replaced. The prepared mutant enzyme genes coding for mutant acid phosphatases and the mutation sites thereof are shown in Table 13. The amino acid residues of the mutation sites represents the amino acid residues in the amino acid sequence shown in SEQ ID NO: 10.

Escherichia coli JM109/pENP180, Escherichia coli JM109/pENP320, Escherichia coli JM109/pENP340, Escherichia coli JM109/pENP410, Escherichia coli JM109/pENP510 and Escherichia coli JM109/pENP520, which were introduced with a plasmid containing each mutant acid phosphatase gene, were each inoculated to 50 ml of L medium containing 100 pg/ml of ampicillin and 1 mM of IPTG, and cultured at 37° C. for 16 hours. The cells were suspended in 50 mL of 100 mM potassium phosphate buffer (pH 7.0), and disrupted by sonication at 4° C. for 20 minutes. The cells were collected from each medium by centrifugation, and washed once with physiological saline. The insoluble fraction in the sonicated suspension was removed by centrifugation to prepare a cell free extract. Km values for inosine and guanosine in the transphosphorylation reaction were measured by using each cell free extract.

The measurement of the transphosphorylation activity for a nucleoside was performed under the following conditions by using inosine and guanosine as substrates. The reaction was performed in a reaction mixture (1 ml) containing various concentrations of inosine or guanosine, 100 μmol/ml of sodium pyrophosphate, 100 μmol/ml of sodium acetate buffer solution (pH 4.0) and each enzyme at pH 4.0 and 30° C. for 10 minutes. After the reaction was stopped by adding 200 μl of 2 N hydrochloric acid, the precipitates were removed by centrifugation, and 5′-inosinic acid or 5′guanylic acid produced by the transphosphorylation reaction was quantified. Inosine, guanosine, 5′inosinic acid and 5′-guanylic acid were analyzed by high performance liquid chromatography (HPLC) under the same conditions as Example 21.

The transphosphorylation activity was measured by changing the concentration of inosine or guanosine in the reaction conditions using the aforementioned composition, and rate constants of inosine and guanosine in the transphosphorylation reaction were determined by Hanes-Woolf plot (Biochem. J., 26, 1406 (1932)). The results are shown in Table 14. As for the Km values of the mutant enzymes produced in the examples, the Km values for guanosine markedly decreased, and thus it was revealed that the affinity for guanosine was improved. Moreover, the Km values for inosine were also markedly reduced in the four kinds of mutant enzymes other than the mutant enzyme encoded by pENP520.

TABLE 13 Plasmid Primer introduced used for intro- Mutation point and with duction of amino acid Plasmid mutation mutation replacement PENP180 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 72G(GGC) → D(GAC) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 151I(ATC) → T(ACC) pENP200 pENP130 MUT180, MUT181 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 72G(GGC) → D(GAC) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 151T(ACC) → S(TCC) pENP300 pENP200 MUT300, MUT301 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 72G(GGC) → D(GAC) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 151I(ATC) → T(ACC) 149T(ACC) → S(TCC) 151T(ACC) → S(TCC) pENP310 pENP300 MUT310, MUT311 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 70A(GCC) → V(GTT) 72G(GGC) → D(GAC) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 151I(ATC) → T(ACC) 149T(ACC) → S(TCC) 151T(ACC) → S(TCC) pENP320 pENP310 MUT320, MUT321 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 70A(GCC) → V(GTT) 72G(GGC) → D(GAC) 102E(GAG) → L(CTG) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 149T(ACC) → S(TCC) 151T(ACC) → S(TCC) pENP330 pENP300 MUT330, MUT331 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 70A(GCC) → M(ATG) 72G(GGC) → D(GAC) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 149T(ACC) → S(TCC) 151T(ACC) → S(TCC) pENP340 pENP330 MUT340, MUT341 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 70A(GCC) → V(GTT) 72G(GGC) → D(GAC) 102E(GAG) → Q(CAG) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 149T(ACC) → S(TCC) 151T(ACC) → S(TCC) pENP400 pENP200 MUT400, MUT401 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 72G(GGC) → D(GAC) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 149T(ACC) → A(GCT) 151T(ACC) → S(TCC) pENP410 pENP400 MUT310, MUT311 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 70A(GCC) → V(GTT) 72G(GGC) → D(GAC) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 149T(ACC) → A(GCT) 151T(ACC) → S(TCC) pENP500 pENP200 MUT500, MUT501 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 72G(GGC) → D(GAC) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 149T(ACC) → G(GGC) 151T(ACC) → S(TCC) pENP510 pENP500 MUT510, MUT511 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 70A(GCC) → E(GAA) 72G(GGC) → D(GAC) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 149T(ACC) → G(GGC) 151T(ACC) → S(TCC) pENP520 pENP500 MUT520, MUT521 61L(CTG) → Q(CAG) 63A(GCT) → Q(CAG) 64E(GAA) → A(GCA) 67N(AAC) → D(GAC) 69S(AGC) → A(GCC) 70A(GCC) → K(AAA) 72G(GGC) → D(GAC) 133T(ACC) → K(AAA) 134E(GAG) → D(GAC) 149T(ACC) → G(GGC) 151T(ACC) → S(TCC)

TABLE 14 Km value Relative Km value for activity when for Relative activity inosine inosine is used guanosine when guanosine is (mM) as substrate (mM) used as substrate pENP180 40 1.0 40 1.0 pENP320 19 1.9 4.6 1.5 pENP340 19 1.4 5.1 1.3 pENP410 18 1.0 4.9 0.70 pENP510 17 0.55 4.0 0.39 pENP520 46 0.63 4.4 0.21

EXAMPLE 23 Transphosphorylation Reaction of Guanosine by E. coli Transformed with a Plasmid Containing Gene for Novel Mutant Acid Phosphatase Derived from Enterobacter Aerogenes IFO12010 Having Improved Affinity for Guanosine

Escherichia coli JM109/pENP180, Escherichia coli JM109/pENP320, Escherichia coli JM109/pENP340, Escherichia coli JM109/pENP410, Escherichia coli JM109/pENP510 and Escherichia coli JM109/pENP520, which were transformed with a plasmid containing each mutant acid phosphatase gene, were inoculated to 50 ml of L medium containing 100 μg/ml of ampicillin and 1 mM of IPTG, and cultured at 37° C. for 16 hours.

10 g/dl of pyrophosphoric acid and guanosine crystals were made into slurry in water, and subjected grinding treatment by a grinding mill (DYNO-MILL produced by WAB, Switzerland). 6.6 g/dl of the obtained guanosine was dissolved in 100 mM acetate buffer (pH 4.5), added with cells of each strain in an amount of 100 mg/dl in terms of dry cell weight, and allowed to react at 35° C. for 12 hours while pH was maintained at 4.5. The amounts of produced 5′-guanylic acid are shown in Table 15. As shown in the table, all of the strains introduced with a mutant enzyme showed improved productivity compared with the parent strain, Escherichia coli JM109/pENP180, and produced and accumulated 5′-guanylic acid with high yield.

TABLE 15 Produced 5′-guanylic Strain acid (g/dl) Escherichia coli JM109/pENP180 9.90 Escherichia coli JM109/pENP320 10.4 Escherichia coli JM109/pENP340 10.2 Escherichia coli JM109/pENP410 11.1 Escherichia coli JM109/pENP510 11.0 Escherichia coli JM109/pENP520 10.5

INDUSTRIAL APPLICABILITY

As explained above in detail, the present invention provides mutant nucleoside-5′-phosphate producing enzymes of which nucleoside-5′-phosphate producing ability is improved, and methods for producing them. The present invention further provides genes coding for the aforementioned mutant enzymes, recombinant DNA containing the genes and microorganisms that harbor the recombinant DNA, which are useful for a method for producing nucleoside-5′-phosphate.

Further, novel three-dimensional structures of proteins were successfully elucidated by X-ray crystallography techniques.

The mutant nucleoside-5′-phosphate producing enzymes of the present invention can be used for the production of nucleoside-5′-phosphates useful as seasonings, drugs, raw materials therefor and so forth. 

1. An isolated mutant acid phosphatase with improved nucleoside-5′-phosphate producing ability relative to the wild type enzyme, wherein the enzyme has the amino acid sequence of SEQ ID NO: 125 wherein said amino acid sequence contains substitution of at least one amino acid residue selected from the group consisting of Leu-14, Leu-61, Ala-63, Glu-64, Asn-67, Ser-69, Ala-70, Gly-71, Gly-72, Ile-101, Glu-102, Thr-133, Glu-134, Leu-138, Thr-149, and Ile-151.
 2. A gene coding for a mutant acid phosphatase according to claim
 1. 3. A recombinant DNA, which contains the gene according to claim
 2. 4. A microorganism comprising the recombinant DNA according to claim
 3. 5. A method for producing a nucleoside-5′ phosphate, which comprises: reacting a nucleoside and a phosphate donor in the presence of a microorganism according to claim 4 to produce nucleoside-5′-phosphate and collecting the nucleoside-5′-phosphate, wherein said phosphate donor is a phosphoric acid ester compound, or a salt thereof, selected from the group consisting of polyphosphoric acid, phenyl phosphate, acetyl phosphate, and carbamyl phosphate.
 6. The method according to claim 5, wherein said reacting is at a pH ranging from 3.0–5.5.
 7. A microorganism comprising the gene according to claim
 2. 8. A method for producing a nucleoside-5′-phosphate, which comprises: reacting a nucleoside and a phosphate donor in the presence of a microorganism according to claim 7 to produce nucleoside-5′-phosphate and collecting the nucleoside-5′-phosphate, wherein said phosphate donor is a phosphoric acid ester compound, or a salt thereof, selected from the group consisting of polyphosphoric acid, phenyl phosphate, acetyl phosphate, and carbamyl phosphate.
 9. The method according to claim 8, wherein said reacting is at a pH ranging from 3.0–5.5.
 10. A method for producing a nucleoside-5′-phosphate, which comprises: reacting a nucleoside and a phosphate donor in the presence of a mutant acid phosphatase according to claim 1 to produce nucleoside-5′-phosphate and collecting the nucleoside-5′-phosphate, wherein said phosphate donor is a phosphoric acid ester compound, or a salt thereof, selected from the group consisting of polyphosphoric acid, phenyl phosphate, acetyl phosphate, and carbonyl phosphate.
 11. The method according to claim 10, wherein said reacting is at a pH ranging from 3.0–5.5.
 12. An isolated mutant acid phosphatase having an amino acid sequence of SEQ ID NO: 125 which contains a series of replacements, wherein said replacements are selected from the group consisting of: (a) Leu-61 with a glutamine residue, Ala-63 with a glutamine residue, Glu-64 with an alanine residue, Asn-67 with an aspartic acid residue, Ser-69 with an alanine residue, Ala-70 with a valine residue, Gly-72 with an aspartic acid residue, Glu-102 with a leucine residue, Thr-133 with a lysine residue, Glu-134 with an aspartic acid residue, Thr-149 with a serine residue and Ile-151 with a serine residue; (b) replacements of Leu-61 with a glutamine residue, Ala-63 with a glutamine residue, Glu-64 with an alanine residue, Asn-67 with an aspartic acid residue, Ser-69 with an alanine residue, Ala-70 with a valine residue, Gly-72 with an aspartic acid residue, Thr-133 with a lysine residue, Glu-134 with an aspartic acid residue, Thr-149 with a alanine residue and Ile-151 with a serine residue; (c) replacements of Leu-61 with a glutamine residue, Ala-63 with a glutamine residue, Glu-64 with an alanine residue, Asn-67 with an aspartic acid residue, Ser-69 with an alanine residue, Ala-70 with a glutamic acid residue, Gly-72 with an aspartic acid residue, Thr-133 with a lysine residue, Glu-134 with an aspartic acid residue, Thr-149 with a glycine residue and Ile-151 with a serine residue; (d) replacements of Leu-61 with a glutamine residue, Ala-63 with a glutamine residue, Glu-64 with an alanine residue, Asn-67 with an aspartic acid residue, Ser-69 with an alanine residue, Ala-70 with a lysine residue, Gly-72 with an aspartic acid residue, Thr-133 with a lysine residue, Glu-134 with an aspartic acid residue, Thr-149 with a glycine residue and Ile-151 with a serine residue; and (e) replacements of Leu-61 with a glutamine residue, Ala-63 with a glutamine residue, Glu-64 with an alanine residue, Asn-67 with an aspartic acid residue, Ser-69 with an alanine residue, Ala-70 with a methionine residue, Gly-72 with an aspartic acid residue, Glu-102 with a glutamine residue, Thr-133 with a lysine residue, Glu-134 with an aspartic acid residue, Thr-149 with a serine residue and Ile-151 with a serine residue.
 13. A method for producing a nucleoside-5′-phosphate, which comprises: reacting a nucleoside and a phosphate donor in the presence of a mutant acid phosphatase according to claim 12 to produce nucleoside-5′-phosphate and collecting the nucleoside-5′-phosphate, wherein said phosphate donor is a phosphoric acid ester compound, or a salt thereof, selected from the group consisting of polyphosphoric acid, phenyl phosphate, acetyl phosphate, and carbamyl phosphate.
 14. The method according to claim 13, wherein said reacting is at a pH ranging from 3.0–5.5.
 15. A gene coding for a mutant acid phosphatase according to claim
 12. 16. A microorganism comprising the gene according to claim
 15. 17. A method for producing a nucleoside-5′-phosphate, which comprises: reacting a nucleoside and a phosphate donor in the presence of a microorganism according to claim 16 to produce nucleoside-5′-phosphate and collecting the nucleoside-5′-phosphate, wherein said phosphate donor is a phosphoric acid ester compound, or a salt thereof, selected from the group consisting of polyphosphoric acid, phenyl phosphate, acetyl phosphate, and carbamyl phosphate.
 18. The method according to claim 17, wherein said reacting is at a pH ranging from 3.0–5.5.
 19. A recombinant DNA, which contains the gene according to claim
 15. 20. A microorganism comprising the recombinant DNA according to claim
 19. 21. A method for producing a nucleoside-5′-phosphate, which comprises: reacting a nucleoside and a phosphate donor in the presence of a microorganism according to claim 20 to produce nucleoside-5′-phosphate and collecting the nucleoside-5′-phosphate, wherein said phosphate donor is a phosphoric acid ester compound, or a salt thereof, selected from the group consisting of polyphosphoric acid, phenyl phosphate, acetyl phosphate, and carbonyl phosphate.
 22. The method according to claim 21, wherein said reacting is at a pH ranging from 3.0–5.5.
 23. A crystal of an acid phosphatase having an amino acid sequence of SEQ ID NO: 124, which has a space group P6₃22 of a hexagonal system.
 24. A crystal of a mutant enzyme acid phosphatase having an amino acid sequence of SEQ ID NO: 124, wherein the Gly-74 is replaced with an aspartic acid residue and Ile-153 is replaced with a serine residue, which has a space group P2₁2₁2₁ of a rhombic system.
 25. A crystal of complex of an acid phosphatase having an amino acid sequence of SEQ ID NO: 124 and molybdate, which has a space group P3₁21 of a trigonal system. 